Advancing Downstream Research Starts with Isolating High-Quality Analytes

LM_ThermoFisherScientific_Compiled_24Advancing downstream research starts with isolating high-quality analytesAutomated sample prep solutions exhibit increased consistency and reproducibility in dataAutomated sample prep solutions deliver greater speed and reproducibility for biomedicaland multiomics researchNext-generation sequencing (NGS), quantitative reverse transcription PCR (qRT-PCR), and digital PCR are powerful tools for analyzing genetic material and circulating biomarkers, facilitating research in oncology, microbiomes, pharmacogenomics, infectious and autoimmune diseases, and multiomic viral studies. However, their success hinges on proper sample preparation.Biomedical and multiomics research depends on carefully isolated genetic material (DNA and RNA) and circulating biomarkers like circulating tumor cells (CTCs). Ensuring quality findings requires consistent and reproducible sample preparation. Traditional methods can be lengthy, cumbersome, and involve hazardous chemicals, but robust, high-throughput commercial solutions simplify and accelerate the process.Automated sample purification instruments using magnetic bead-based technology moves the beads instead of the liquids. This results in higher purity because no non-target organic matter or contaminates from liquid reagents are transferred from well to well. The analyteof interest binds to the bead - even from challenging sample time including Formalin-fixed paraffin-embedded samples.The field of biomedical and multiomics research is experiencing rapid advancements, primarily attributed to the meticulous and thorough preparation of samples. This process is crucial in generating data that is of high quality and significance. To assist researchers in this endeavor, Thermo Fisher Scientific has compiled a series of application notes that provide a comprehensive overview of the KingFisher automated sample purification instrument. These notes cover a wide range of research areas, including microbiome research, multiomic viral research, and cancer research, showcasing the instrument’s versatility. By exploring these application notes and carefully reviewing the accompanying data, researchers can gain valuable insights into best practices and emerging techniques. This knowledge will enable them to optimize their workflows and achieve more reliable and accurate results in their research endeavors.2ContentsMicrobiomeFecal microbiome diversity: family, diet, probiotics, and pregnancyas associated factors (2022) 4Investigating microbiomes in various sample types withthe MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (2022) 10Metagenomic analysis of the human microbiome witha new MagMAX kit automated on a KingFisher platform (2019) 14Multiomics Viral ResearchRapid bead-based viral enrichment for multiomic viral research 20Cancer ResearchIsolation of circulating tumor cells using Dynabeads magnetic beads 25Extraction and analysis of circulating cell-free DNA from plasma samples for lung cancer research 30A complete next-generation sequencing workflow for circulatingcell-free DNA isolation and analysis 34Genomic DNA extraction from bone marrow aspirates and peripheral blood mononuclear cells 42Versatile solutions for human leukocyte antigen testing withperipheral blood 46Application note | MagMAX Microbiome Ultra Nucleic Acid Isolation Kit‌Nucleic acid isolationFecal microbiome diversity: family, diet, probiotics, and pregnancy as associated factorsSummaryIntroductionThe human gastrointestinal tract contains the most diverse microbial community in the body. Factors that can affect the composition of the gut microbiota include the externalenvironment, genetics, lifestyle, nutrition, health status, and age. Although gut microbiomes differ in diversity across individuals, family members living in the same household are often observed to have more similarities between their microbiota than unrelated individuals [1]. Familial similarities are usually attributed toshared environmental influences such as dietary preference, a powerful shaper of microbiome composition [2,3]. There isgrowing evidence that close social relationships correlate with the composition of the human gut microbiota [4]. Furthermore, the gut microbiome is impacted by diet [3] and the use of probiotics [5]. Pregnancy has also been shown to affect the microbiota of both mother and child in diverse ways. The microbiota governs the infant’s immune, allergic, and metabolic responses—eveninto adulthood. Delivery method, exposure to antibiotics, and breastfeeding have a significant impact on the gut microbiome and on the overall health of the infant [6]. Here we investigate four case studies that focus on different factors affecting the composition of the gut microbiome.The use of a consistent methodology is an important component in researching the role of gut microbiota in health and disease.Reliable and reproducible extraction of nucleic acids from the gut microbiome is essential to avoid the introduction of biases during sample preparation. Here we report on the utility of the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit for studying the gut microbiome using total nucleic acids extracted fromfecal samples. Microbial diversity was evaluated by 16S rRNA sequencing using the Ion GeneStudio™ S5 System. We highlight the use of the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit in the effective analysis of the microbial community from a single family (father, mother, and son); the microbial community changes associated with diet and probiotics usage; and the distinct changes in microbial profiles that occur throughout pregnancy and the postpartum period. Furthermore, we demonstrate that various workflow options of the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit show equivalent performance when compared to other similar kits available on the current market.Materials and methodsTotal nucleic acid extraction using the MagMAX Microbiome Ultra Nucleic Acid Isolation KitHuman fecal samples were obtained from donors, and isolations of total nucleic acid were performed in duplicate using the standard protocol for the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. For benchmarking experiments, fecal samples were collected from two donors and processed using various workflow options from the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit with the KingFisher Flex Purification System. These options included bead-beating in tubes for 10 minutes, plate bead-beating for 2 minutes, plate bead-beating for 20 minutes, and tube bead-beating for 10 minutes and performing a manual nucleic acid extraction. Similar kits from QIAGEN included spin column and magnetic bead–based options (DNeasy™ PowerSoil™ Kit and MagAttract™ PowerSoil™ DNA Kit, respectively), and isolations were performed following each kit’s standard protocol. For evaluating the microbial profiles of individual family members, fecal samples were collected from a father, mother, and son living in the same household but following different diets.Probiotics case study: Fecal samples were collected from an individual 1 week before probiotic usage to create a baseline. Additional fecal samples were collected every week during probiotic usage for 8 weeks and at week 12, which marked the end of probiotic consumption.Diet case study: Samples were collected from an individual before following the diet schedule to create a baseline and at 16 weeks after following the vegetarian dietary changes recommended by a nutritionist.Pregnancy and postpartum case study: Samples were collected from the pregnant donor at 5 timepoints: at 23 weeks gestation, at 33 weeks gestation, at 37 weeks gestation, at 3 weeks postpartum, and at 8 weeks postpartum.An input of 100 mg of feces in 400 µL of lysis buffer was used for all extractions, for a total extraction volume of 400 µL. Extractions were done in duplicate, and the final samples were eluted in200 µL of elution solution. Total nucleic acid was isolated in an automated fashion using the Thermo Scientific™ KingFisher™ Flex Magnetic Particle Processor with 96 Deep-well Head. 16S rRNA sequencing was performed on total nucleic acid using the Ion GeneStudio S5 System as shown in Figure 1. The RStudio™ program was used to generate heat maps.ResultsComparison of other kits to the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit using different workflow optionsThe MagMAX Microbiome Ultra Nucleic Acid Isolation Kit can be used to efficiently isolate total nucleic acid from human fecal samples using its various recommended workflows. We compared the microbiome profiles generated from total nucleicacid isolated using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit with other kits available on the market (Figure 2).Although the total microbiome profiles remained consistent across all extraction types, there was a greater relative percent abundance of microbial diversity detected by workflows from the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit compared to the spin-column and magnetic bead–based kits (DNeasy PowerSoil Kit and MagAttract PowerSoil DNA Kit, respectively) from QIAGEN. For instance, a whole metagenome profile showed a greater abundance of Prevotellaceae in donor 1 using any of the four workflows tested for the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. The spin-column extraction usingthe DNeasy PowerSoil Kit showed relatively low abundance for Prevotallaceae, while there was no detection of this family with the MagAttract PowerSoil DNA Kit. Bacteroidaceae waspredominantly higher in donor 2 with very little to no detection of Prevotellaceae using any of the compared extraction workflows, suggesting that there is variation between donors for this family of bacteria.Comparison of the microbiome profiles between family members: a case studyFecal samples were collected from a single family living in one household in which individuals had different dietary habits.Total nucleic acid was isolated using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. Members of the family—father, mother, and son—showed strikingly similar microbial diversity for a few species of Bacteroides, Eubacterium, and Ruminococcus (Figure 3). However, significant differences were apparent between individuals. Prevotella strains are associated withplant-rich diets [7], suggesting that the significantly higher abundance of Prevotella copri from donor 1 (the father) is attributable to his vegetarianism.Multiple sample typesMagMAX Microbiome Ultra Nucleic Acid Isolation KitKingFisher Flex Magnetic Particle ProcessorIon 16SIon GeneStudioMetagenomics Kit S5 SystemIon Reporter SoftwareSample Primer Primerpool 1 pool 2orHypervariable regions Primer pool 1: V2, V4, V8Primer pool 2: V3, V6, V7, V9Sample collectionBead-based cell lysisExtractionAmplificationSequencingAnalysisFigure 1. End-to-end workflow for 16S rRNA sequencing. The Ion 16S™ Metagenomics Kit and the Ion Plus Fragment Library Kit were used to synthesize 16S rRNA libraries. The barcoded libraries were pooled and templated on the Ion Chef™ Instrument followed by sequencing on the Ion GeneStudio S5 System. Automated analysis, annotation, and taxonomic assignments were performed using Ion Reporter™ Software.High MediumAlistipes finegoldii Alistipes indistinctus Alistipes onderdonkii Alistipes putredinisBifidobacteriaceaeLowAllisonella histaminiformans Bacteroides caccae Bacteroides fragilis Bacteroides massiliensis Bacteroides ovatus Bacteroides stercoris Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides vulgatus Bacteroides xylanisolvens Bacteroides cellulosilyticus Bacteroides faecichinchillae Bacteroides intestinalis Barnesiella intestinihominis Bifidobacterium catenulatum Bifidobacterium longum Bifidobacterium adolescentis Bifidobacterium breveBifidobacterium pseudocatenulatumBifidobacterium ruminantium Bilophila wadsworthia Blautia faecisBlautia luti Blautia producta Blautia wexleraeRikenellaceaeRuminococcaceaeClostridium spiroforme Collinsella aerofaciens Coprococcus catus Coprococcus comes Coprococcus eutactus Dialister invisusCoriobacteriaceaeDesulfovibrionaceaeSutterellaceaeDorea formicigenerans Dorea longicatena Eggerthella lenta Enterococcus faecalis Eubacterium eligens Eubacterium hallii Eubacterium hadrum Eubacterium ventriosum Eubacterium ramulus Eubacterium rectale Faecalibacterium prausnitzii Flavonifractor plautii Gemmiger formicilis Haemophilus parainfluenzae Holdemania massiliensisLachnoclostridium clostridioformePrevotellaceaeLactobacillus rogosae Mannheimia varigena Odoribacter splanchnicus Parabacteroides distasonis Parabacteroides merdae Parasutterella excrementihominis Phascolarctobacterium faecium Prevotella copriMagMAX Microbiome kit: tube 10 min MagMAX Microbiome kit: HT 2 min MagMAX Microbiome kit: HT 20 min MagMAX Microbiome kit: manual 10 min QIAGEN DNeasy PowerSoil KitQIAGEN MagAttract PowerSoil DNA KitRoseburia faecis Roseburia intestinalis Roseburia hominis Roseburia inulinivorans Ruminococcus faecis Ruminococcus gauvreauii Ruminococcus gnavus Ruminococcus torques Ruminococcus bromii Senegalimassilia anaerobiaStreptococcus thermophilus Streptococcus salivarius Sutterella wadsworthensisBacteroidaceaeClostridiaceaeVellionellaceaeLachnospiraceaePorphyromonadaceaeRikenellaceaeRuminococcaceaeCoriobacteriaceaeBacteroidaceaeDesulfovibrionaceaeClostridiaceaeSutterellaceaePrevotellaceaeVellionellaceaeLachnospiraceaePorphyromonadaceaeB454035302520151050Abundance (%)BifidobacteriaceaeA454035302520151050MagMAX Microbiome kit: tube 10 min MagMAX Microbiome kit: HT 2 min MagMAX Microbiome kit: HT 20 min MagMAX Microbiome kit: manual 10 min QIAGEN DNeasy PowerSoil KitQIAGEN MagAttract PowerSoil DNA KitAbundance (%)Figure 2. Benchmarking microbiome kit workflows with fecal samples from two donors (A and B). MagMAX Microbiome Ultra Nucleic Acid Isolation Kit workflows show superior performance in terms of relative abundance of diverse bacterial families compared to kits from QIAGEN.Son: replicate 2Son: replicate 1Mother: replicate 2Mother: replicate 1Father: replicate 2Father: replicate 1Figure 3. Microbiome profile within a single family. Fecal samples collected from individuals within a single family were processed using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. 16S rRNA sequencing was accomplished using the Ion Torrent platform. The DNA profile showed a unique microbiome from each individual at the species level.Effects of probiotics on the gut microbiome: a case studyProbiotics intake for 12 weeks was associated with alteration of a donor’s microbiome profile for Firmicutes and Bacteroidetes. Fecal samples were collected before starting the probiotic course, then at every week for 8 weeks and at week 12 of probiotics usage. Total nucleic acid was isolated using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. 16S rRNA sequencing was performed to determine the microbiome diversity associated with probiotic usage (Figure 4). There was no significant difference over the course of the study in levels of Proteobacteria. Levels of Firmicutes were high at baseline but they decreased with usage of probiotics. Bacteroidetes were at medium levels before probiotics usage but were elevated after probiotics intake. The Firmicutes/Bacteroidetes (F/B) ratio iswidely accepted to influence normal gut homeostasis. Increased or decreased F/B ratios are considered dysbiosis, which is associated with the development of obesity or inflammatory bowel disease (IBD), respectively [8]. In this case study, the consumption of probiotic preparations containing Lactobacillus in combination with Bifidobacterium led to a slight reduction in the F/B ratio as early as week 1. The early reduction in the F/B ratio suggests that the probiotic usage contributed to that reduction and to reversal of gut dysbiosis, which remained consistent throughout 12 weeks of probiotic consumption. However, the levels of Actinobacteria started at medium-to-low at baseline and decreased over time. Consistent with the case study, probiotic preparations containing Bifidobacteria are thought to contribute to a reduction in Actinobacteria that results in gut homeostasis; however, larger clinical studies are needed to confirm these encouraging results [9].High Medium LowProteobacteria Firmicutes Bacteroidetes ActinobacteriaWeek 12Week 8Week 7Week 6Week 5Week 4Week 3Week 2Week 1Before probioticsFigure 4. Effects of probiotics on the human gut microbiome. Fecal samples were collected from a donor before probiotic usage and forup to 12 weeks during probiotic consumption. Total nucleic acid was isolated using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. 16S rRNA sequencing using the Ion GeneStudio S5 System showed a distinct microbiome profile shift for Firmicutes and Bacteroidetes levels over 12 weeks (blue arrows).Effects of diet on gut microbiome: a case studyThe MagMAX Microbiome Ultra Nucleic Acid Isolation Kit efficiently differentiated the intestinal microbiome to the species level for an individual who followed a strict vegetarian diet (Figure 3). Figure 5 shows that a vegetarian diet affects the relative abundance of certain species and significantly alters the gut microbial diversity relative to a non-vegetarian diet. This is consistent with studies that have shown that a vegetarian diet results in an increase in abundance of bacteria such as Clostridium that ferment dietary fiber [10-12], a reduction ofthe proportion of carbohydrate-metabolizing Bacteroidessformed valuesHigh MediumLow[13,14], and a decrease in the abundance of butyrate-producing Bifidobacterium [3]. Accordingly, several species belonging to the bile-tolerant Alistipes, which has previously been associated with a meat-rich diet [3], showed a lower abundance after the strict vegetarian diet was followed.Akkermansia muciniphila Alistipes finegoldii Alistipes indistinctus Alistipes onderdonkii Alistipes putredinis Alistipes timonensisAllisonella histaminiformans Bacteroides clarus Bacteroides faecichinchillae Bacteroides fragilis Bacteroides intestinalis Bacteroides nordii Bacteroides oleiciplenus Bacteroides ovatus Bacteroides stercorirosoris Bacteroides stercoris Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides vulgatus Bacteroides xylanisolvens Bifidobacterium adolescentis Bifidobacterium catenulatumBifidobacterium kashiwanohense Bifidobacterium longum Bifidobacterium ruminantium Bilophila wadsworthiaBlautia producta Blautia wexlerae Clostridium aldenense Clostridium innocuum Clostridium lavalense Clostridium leptum Coprococcus comes Coprococcus eutactusCorynebacterium tuberculostearicum Dialister invisusDielma fastidiosa Dorea formicigenerans Dorea longicatena Eubacterium eligens Eubacterium hadrum Eubacterium hallii Eubacterium rectaleEubacterium ventriosum Faecalibacterium prausnitzii Flavonifractor plautii Gemmiger formicilis Haemophilus parainfluenzae Holdemania filiformisLachnoclostridium clostridioforme Lactobacillus rogosae Mannheimia varigenaOdoribacter splanchnicus Oxalobacter formigenes Parabacteroides distasonis Parabacteroides goldsteinii Parabacteroides merdae Parasutterella excrementihominis Pseudobutyrivibrio ruminis Roseburia faecisRoseburia hominis Roseburia intestinalis Roseburia inulinivorans Ruminococcus bromii Ruminococcus faecis Ruminococcus gauvreauii Ruminococcus gnavus Ruminococcus lactaris Streptococcus salivarius Sutterella wadsworthensis Veillonella rogosae16-week diet No dietFigure 5. Effects of diet on the human gut microbiome. 16S rRNA sequencing results from DNA extracted from fecal samples using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit were analyzed to compare changes in an individual’s microbiome profile following dietary changes that occurred over 16 weeks.Changes in microbiota profile during pregnancy: a case studyFecal samples were collected from the pregnant donor at 5 timepoints: at 23 weeks gestation, at 33 weeks gestation, at 37 weeks gestation, at 3 weeks postpartum, and at 8 weeks postpartum. Figure 6 shows that some bacterial families changed dramatically over the course of pregnancy and some stayed very similar. More research needs to be done to fully understand the cause and effect of gut microbiome changessformed valuesHigh MediumLowAcidaminococcaceae Bacteroidaceae Bifidobacte riaceae Clostridiaceae Coriobacte riaceae Desul fovibrionaceae Enterobacte riaceae Erysipelot richaceae Eubacteriaceae Hyphomicrobiaceae Lachnospi raceae Lactobacillaceae PasteurellaceaePeptostreptococcaceae Porphyromonadaceae PrevotellaceaeRikenellaceae Ruminococcaceae Streptococcaceae Sutterellaceae Veillonellaceaeover time in a pregnancy. Since microbiomes differ significantly in individuals, this study was meant to look at the profile of temporal concentration changes.Week 23 gestationWeek 33 gestationWeek 37 gestationWeek 3 postpartumWeek 8 postpartumFigure 6. Comparison of the microbiome profile during pregnancy (at 23, 33, and 37 weeks of gestation) and postpartum (at 3 and8 weeks).ConclusionTotal nucleic acid from fecal samples can be efficiently isolated using the MagMAX Microbiome Ultra Nucleic Acid IsolationKit for downstream applications. Notably, DNA and RNA can be isolated for comprehensive analyses of the human gut microbiome. In addition, the workflow comparison with othersuppliers shows better performance of the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit in terms of the relative abundance of different microbial families and comparable results in termsof distinguishing microbial profiles. This study showed the equivalent performance of all workflow options explored with the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. This consistent performance enables a researcher to study the microbiome profiles from fecal samples without any isolation biases. In addition, the changes in microbiome profiles between individuals living in the same household who have different dietary habits can be efficiently studied. The relative changes in microbiome profiles following diet and probiotics usage can beeasily differentiated using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. Finally, the changes in microbiota profiles observed during pregnancy and postpartum can be analyzed following nucleic acid isolation with the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit.ReferencesScience 334:105–108. doi:10.1126/science.1208344.ContributorsMadhuri Jasti, Jay Bhandari, Anupriya Gupta,Laura Chapman Mier, Stacie Ferguson, Marie Gonzalez Learn more at For Research Use Only. Not for use in diagnostic procedures. © 2022 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. DNeasy and MagAttract are trademarks of QIAGEN. PowerSoil is a trademark of Mo Bio Laboratories, Inc. RStudio is a trademark of RStudio, PBC. COL35102 0822Application note | MagMAX Microbiome Ultra Nucleic Acid Isolation Kit‌Nucleic acid isolationInvestigating microbiomes in various sample types with the MagMAX Microbiome Ultra Nucleic Acid Isolation KitSummaryIntroductionA microbiome is a collection of genomes in a population of microbes [1]. Distinct populations of microbes reside in various parts of the human body, and knowing where specific types of microorganisms live can provide insight into their functions and the interactions they have with the body. For example, human skin supports a large and diverse population of microbes that can affect skin health [2,3]. Milk provides essential microbesto nursing infants, and human milk has been shown to have a diverse microbiome that is thought to be beneficial [4]. Nucleic acid can be efficiently extracted from soil and stool samples using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit (Cat. No. A42358). In this application note, we demonstrate that it is also effective for extracting microbial nucleic acid from skin swabs, armpit swabs, cow milk, and human milk.Microbiome end-to-end workflowMaterials and methods Sample collectionSkin and armpit swabs were collected in duplicate by swabbing the surfaces of a human hand and armpit, respectively. The armpit swabs were obtained from a single donor who was not wearing deodorant. Human milk was obtained from a single donor 3 weeks, 7 weeks, and 19 weeks after giving birth. All of the human samples were processed in duplicate. USDA-certified organic and conventional whole cow milk samples were also collected and processed in duplicate.Nucleic acid extraction and analysisTo prepare the milk samples for extraction, 400 µL of each sample was added to the bead beating plate and processed in 800 µL of lysis buffer. After removing the shafts from the hand and armpit swabs, each swab was also processed in800 µL of lysis buffer in the bead beating plate. A Bead Ruptor™ 96 homogenizer was used for bead beating, and extraction was performed with the Thermo Scientific™ KingFisher™ Flex Purification System fitted with a 96 deep-well plate. After purification, each isolated nucleic acid sample (DNA and RNA) was eluted in 200 µL of elution solution.Note: One duplicate set of hand swabs and one duplicate set of armpit swabs did not receive proteinase K treatment. This allowed changes in the abundance of gram-positive and gram-negative bacteria to be observed.Multiple sample MagMAXtypesMicrobiome Ultra Nucleic Acid Isolation KitKingFisher instrumentIon 16SIon TorrentMetagenomics Kit sequencingsystemsMy Primer Primer sample pool 1 pool 2Ion Reporter SoftwareorHypervariable regions Primer pool 1: V2, V4, V8Primer pool 2: V3, V6, V7, V9Sample collectionBead-based cell lysisExtractionAmplificationSequencingAnalysisThe Ion Plus™ Fragment Library Kit and the Ion 16S™ Metagenomics Kit were used to synthesize 16S rRNA gene™libraries on the Ion Chef Instrument. The barcoded librariesFigure 1. Sample collection, extraction, library creation, sequencing, and analysis workflow.were pooled and templated on the Ion Chef Instrument, then sequenced on the Ion GeneStudio™ S5 System. Automated analysis, annotation, and taxonomic assignment were performed using Ion Reporter™ Software. The RStudio™ program was used to generate heat maps. The nucleic acid extraction and analysis workflow is illustrated in Figure 1.AcidaminococcaceaeActinomycetaceae 4BacillaceaeBacillales incertae sedis 2BacteroidaceaeBifidobacteriaceae 0CampylobacteraceaeCarnobacteriaceae −2ClostridiaceaeClostridiales Family XI. Incertae Sedis −4CoriobacteriaceaeCorynebacteriaceae −6No_PK_HandsPK_Hands−8Enterobacteriaceae Enterococcaceae Erysipelotrichaceae Eubacteriaceae Flavobacteriaceae Fusobacteriaceae Lachnospiraceae Lactobacillaceae Leptotrichiaceae Micrococcaceae Moraxellaceae Neisseriaceae Oxalobacteraceae Pasteurellaceae Peptoniphilaceae Peptostreptococcaceae Porphyromonadaceae Prevotellaceae Propionibacteriaceae Pseudanabaenaceae Rhodobacteraceae Rikenellaceae Ruminococcaceae Sphingomonadaceae Spirochaetaceae Staphylococcaceae Streptococcaceae Streptomycetaceae Sutterellaceae Synergistaceae VeillonellaceaeAcidaminococcaceaeResultsWe successfully extracted bacterial nucleic acid from skin swabs, sweat on armpit swabs, and milk samples using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. With the isolated nucleic acid, we were able to confirm the presenceof various families of bacteria (Figure 2). We were also able to observe differences between bacterial abundance in various parts of the human body. Staphylococcaceae andCorynebacteriaceae are known to be abundant in moist areasActinomycetaceae 4BacillaceaeBacillales incertae sedis 2BacteroidaceaeBifidobacteriaceae 0CampylobacteraceaeCarnobacteriaceae −2ClostridiaceaeClostridiales Family XI. Incertae Sedis −4CoriobacteriaceaeCorynebacteriaceae −6of the human body. These families are less abundant in drier areas like the hands [5,6]. As shown in Figure 3, samples that were treated with proteinase K contained higher concentrations of nucleic acid from gram-positive bacteria, including Actinomycetaceae, Micrococcaceae, Propionibacteriaceae, and Streptomycetaceae [7-10]. Conversely, the concentrations of nucleic acid from gram-negative bacteria, such as Bacteroidaceae and Porphyromonadaceae, were higher inuntreated samples [11,12].No_PK_ArmpitsNo_PK_Hands−8Enterobacteriaceae Enterococcaceae Erysipelotrichaceae Eubacteriaceae Flavobacteriaceae Fusobacteriaceae Lachnospiraceae Lactobacillaceae Leptotrichiaceae Micrococcaceae Moraxellaceae Neisseriaceae Oxalobacteraceae Pasteurellaceae Peptoniphilaceae Peptostreptococcaceae Porphyromonadaceae Prevotellaceae Propionibacteriaceae Pseudanabaenaceae Rhodobacteraceae Rikenellaceae Ruminococcaceae Sphingomonadaceae Spirochaetaceae Staphylococcaceae Streptococcaceae Streptomycetaceae Sutterellaceae Synergistaceae VeillonellaceaeFigure 2. Abundance of bacteria on hand and armpit swabs without proteinase K (PK) treatment. Each data point is reported as the average of technical duplicates.Figure 3. Abundance of bacteria on hand swabs with and without proteinase K (PK) treatment. Each data point is reported as the average of technical duplicates.We also compared the abundance of microbiota in human breast milk, organic cow milk, and conventional whole cow milk (Figure 4). The microbiomes of human and cow milk differed significantly, and there also were differences between the microbiomes of organic and conventional whole milk.Hyphomicrobiaceae were not present in either organic or conventional cow milk, but they were both present in human milk 7 and 19 weeks after delivery. Ruminococcaceae were less abundant in cow milk as well as human milk during the50−5early postpartum period. However, they were highly abundant in human milk during the later postpartum period. More research is required to understand why the microbiome of human milk changes over time.ConclusionWe have demonstrated the versatility of the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit by extracting nucleic acidfrom gram-negative and gram-positive bacteria in cow milk, human milk, and human hand and armpit swabs. We found that proteinase K treatment could alter the yields of nucleic acid from gram-positive and gram-negative bacteria. We could efficiently obtain high yields of nucleic acid from both high- and low-abundance species. The versatility and robustnessof the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit will make it a welcome tool in any laboratory that performs microbiome research.Acidaminococcaceae Actinomycetaceae Alteromonadaceae Bacillaceae Bacteroidaceae Bifidobacteriaceae Burkholderiaceae Carnobacteriaceae Catabacteriaceae Christensenellaceae ClostridiaceaeClostridiales Family XI. Incertae Sedis Coriobacteriaceae Corynebacteriaceae Desulfovibrionaceae Enterobacteriaceae ErysipelotrichaceaeEubacteriaceae Flavobacteriaceae Fusobacteriaceae Gracilibacteraceae Halomonadaceae Hyphomicrobiaceae Intrasporangiaceae Lachnospiraceae Lactobacillaceae Micrococcaceae Moraxellaceae Mycoplasmataceae Oceanospirillaceae Oscillospiraceae Pasteurellaceae Porphyromonadaceae Prevotellaceae Propionibacteriaceae Pseudomonadaceae Rhizobiaceae Rhodospirillaceae Rikenellaceae Ruminococcaceae Shewanellaceae Spirochaetaceae Staphylococcaceae Streptococcaceae Succinivibrionaceae Sutterellaceae Veillonellaceae VerrucomicrobiaceaeBreast_Milk_19WeeksAfterDelivery Breast_Milk_7WeeksAfterDelivery Breast_Milk_3WeeksAfterDelivery Organic_MilkWhole_MilkFigure 4. Heat maps of bacteria in organic and conventional whole cow milk and human breast milk collected at 3 different time points.References1 Kb PlusDNA Ladder1 2 1 212521Donor 1Donor 2DNARNA7060Total yield (µg)5040Figure 2. Quality of total microbial nucleic acid purified from feces. For each donor, duplicate 1 µg samples of total nucleic acid were run on a 1% agarose gel. Clear bands of both genomic DNA and RNA can be seen. The Invitrogen™ 1 Kb Plus DNA Ladder was used for size determination.Note that since this is not a denaturing gel, apparent sizes of the RNA differ from actual sizes.1.961.8464.545.73020100Donor 1 Donor 2A260/A280Figure 1. Total nucleic acid yields from 100 mg fecal samples. Samples from two donors were processed using the MagMAX Microbiome kit. The KingFisher Flex Magnetic Particle Processor with 96 Deep-Well Head was used for isolation, with an up-front bead beating step. A260/A280 values are indicated.TaqMan qPCR Assays were performed for Firmicutes, Bacteroidetes, and E. coli using total nucleic acid isolated from fecal, saliva, and urine samples. All of these bacteria are routinely found in the human body, though E. coli levels are typically low. By probing for these two phyla and E. coli, a broad range of coverage was tested. Asexpected, fecal samples contained high levels of Firmicutes and Bacteroidetes (Figure 3A), whereas urine and saliva samples contained high levels of Firmicutes (Figures 3Band C). E. coli abundance was lower, but in the detectable range, for all sample types tested (Figures 3A, B, and C). A TaqMan Assay with Applied Biosystems™ Xeno™ DNA and RNA controls showed no inhibition from the total nucleic acid preparations (data not shown), confirming that the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit enables isolation of high-quality, inhibitor-free nucleic acid, even from the most challenging samples.A Fecal26.617.816.122.917.616.63025Mean Ct20151050Donor 1 Donor 2B Urine29.0428.9712.96.835 32.17 32.463025Mean Ct20151050Donor 1 Donor 2SalivaC34.133.729.828.47.610.0403530Mean Ct2520151050Donor 1 Donor 2Firmicutes Bacteroidetes E. coliFigure 3. qPCR analysis of DNA purified from (A) fecal, (B) urine, and (C) saliva samples of two donors, with the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit. TaqMan Assays were utilized for one category of gram-positive (Firmicutes) and two categories of gram-negative (Bacteroidetes and E. coli) bacteria. The total nucleic acid samples were diluted 1:100 for the Firmicutes and Bacteroidetes assays, whereas the input was not diluted for the E. coli assay. TaqMan Fast Advanced Master Mix was used under fast cycling conditions.Shotgun metagenomics sequencing of total nucleic acid isolated from feces, urine, and salivaTotal nucleic acid from fecal, urine, and saliva samples from two donors was isolated and sequenced; Figure 4 shows the metagenomics sequencing data as abundance heat maps for all fecal, urine, and saliva samples. The abundance heat maps shown here do not represent the full microbiome profile for each sample type. The species-level whole-genome profiles are shown in Figure 5. Note that the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit can isolate viral total nucleic acid in addition to bacterial (shown in urine and saliva profiles in Figure 4). Metagenomics profiles for feces and saliva have only Prevotella speciesas a common bacterial target, whereas urine and saliva metagenomics profiles share the same viral targets.Metagenomics sequencing of fecal nucleic acid from donor 1 (zr2132.1) shows high abundance of Prevotella,Faecalibacterium, Roseburia, Bacteroides, Alistipes, and others, and from donor 2 (zr2132.2) shows high abundance of several Bacteroides, Faecalibacterium, Bifidobacterium,Parabacteroides, and others. All samples were mapped to a human database using the program Centrifuge (data not shown), and the data demonstrated that the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit enriches forisolation of bacterial targets up to 99%, and human targets were detected at a very low level (0.06–0.45%) in thefecal samples.A whole-metagenome profile at the species level shows multiple bacterial and viral targets detected in the profile that are specific to a given sample type. Fecal samples from both donors show high amounts of Bacteroidetes,Firmicutes, Actinobacteria, and Proteobacteria. The fecal sample from donor 1 shows Prevotella copri, several Bacteroides species, Rothia, Faecalibacterium,Sutterella, and other species as highly abundant, whereas the fecal sample from donor 2 shows high abundanceof Bacteroides, Bifidobacterium, Faecalibacterium, Rothia, Sutterella, and other species. Urine samples from both donors have high abundance of Lactobacillus, Streptococcus, Bacteroides, Ruminococcus, andBifidobacterium, Pseudomonas phages, and other species.Saliva samples from both donors show high abundance of Streptococcus mitis, other Streptococcus species, Granulicatella, Neisseria, Prevotella, and other species (Figure 5).ConclusionWe have developed an automated, faster workflow for isolation of high-quality, inhibitor-free DNA and RNA from human stool samples and other sample types, using the new MagMAX Microbiome Ultra Nucleic Acid Isolation kit. Whole-genome sequencing of samples from two donors showed the total profile of microorganisms in feces, urine, and saliva. The workflow that we developed to harness the power of microbiome research enables fast generation of metagenomic data for bacterial communities residing within human body, which can be used as diagnostic biomarkers for certain diseases, and potentially pave the path for future microbiome therapeutics.10-1100101Shotgun sequencing data at species levelzr2132.1zr2132.2zr2132.3zr2132.4zr2132.5zr2132.6FecalUrineSalivaFigure 5. Identification of whole-metagenome profiles for fecal, urine, and saliva samples using shotgun metagenomics sequencing.Each color represents a different operational taxonomic unit (OTU) identified per sample.zr2132.1zr2132.2zr2132.3zr2132.4zr2132.5zr2132.6FecalUrineSalivaFigure 4. Shotgun metagenomics sequencing abundance heat maps for fecal, urine, and saliva sample isolations.Shotgun sequencing data at species levelTop 10 abundant species from zr2132.1 (donor 1, feces)Top 10 abundant species from zr2132.2 (donor 2, feces)Prevotella copri Faecalibacterium prausnitzii Bacteroides vulgatus Alistipes putredinis Barnesiella intestinihominis Roseburia intestinalis Bacteroides thetaiotaomicron Bacteroides ovatusTop 10 abundant species from zr2132.3 (donor 1, urine)Sutterella wadsworthensis Bacteroides caccaeBacteroides vulgatus Faecalibacterium prausnitzii Bacteroides uniformis Bacteroides stercoris Alistipes putredinis Eubacterium rectale Ruminococcus bromii Bifidobacterium longum Bacteroides thetaiotaomicron Parabacteroides distasonisTop 10 abundant species from zr2132.5 (donor 1, saliva)Pseudomonas phage F10 Pseudomonas phage B3 Pseudomonas phage Pf1 Pseudomonas aeruginosa Pseudomonas phage DMS3 Halomonas stevensii Unclassified Halomonas Human adenovirus B Unclassified Alcaligenes Varibaculum cambrienseTop 10 abundant species from zr2132.6 (donor 2, saliva)Top 10 abundant species from zr2132.4 (donor 2, urine)Pseudomonas phage F10 Pseudomonas phage B3 Pseudomonas phage Pf1 Pseudomonas aeruginosa Pseudomonas phage DMS3 Lactobacillus crispatus Halomonas stevensii Unclassified Alcaligenes Prevotella bivia Haemophilus parainfluenzaeHaemophilus parainfluenzaeStreptococcus mitis, oralis, and pneumoniae Streptococcus australisPrevotella spp. oral taxon 473 Neisseria flavescens Streptococcus infantis Streptococcus sp. GMD4S Prevotella melaninogenica Veillonella parvulaVeillonella sp. oral taxon 158Prevotella melaninogenica Streptococcus australis Streptococcus parasanguinis Veillonella atypica Haemophilus parainfluenzae Prevotella pallensVeillonella parvulaPorphyromonas sp. oral taxon 279 Streptococcus infantis Streptococcus salivariusReferencesFind out more atthermofisher.com/magmaxmicrobiomeFor Research Use Only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. ZymoBIOMICS is a trademark of Zymo Research Corp. KAPA is a trademarkof Roche Molecular Systems, Inc. TruSeq and HiSeq are trademarks of Illumina, Inc. Python is a trademark of Python Software Foundation. TapeStation is a trademark of Agilent Technologies, Inc. COL32949 0219Application note | Dynabeads Intact Virus Enrichment‌Rapid bead-based viral enrichment for multiomic viral researchOptimizing with Dynabeads Intact Virus EnrichmentKeywordsDynabeads, enrichment, SARS-CoV-2, virus-like particles, western blot, qPCR, plaque assay, viral transport medium, cell culture medium, wastewater, influenza virus A (INFV), H1N1, respiratory syncytial virus, Zika virus, adenovirus, enterovirus, norovirusIn this application note, we show:IntroductionVirus enrichment is an essential method for obtaining viruses in the quantities often required to study their life cycles and pathogenesis. Multiomic study of viruses requires isolation of intact virus particles, often from large volumes and very dilute samples. To obtain sufficient amounts of viruses, stocks of virus can be made by inoculation of cell cultures with a seed virus. The infected cells will release new viral particles into the cell culture medium (CCM) at the end of the viral life cycle. The released viral particles are then extracted from the medium.Enrichment of viruses is challenging because of their size and typical concentrations in samples. Virus enrichment can be a tedious and difficult process, and may result in insufficient yield. Low virus titers can introduce artificial variants, or bias in gene sequences. A range of methods that include different forms of ultracentrifugation, precipitation, and filtration are used to enrich virus particles from cell culture supernatants; these methods require expensive instruments, are very time-consuming, and may result in low yields.Fast and simple virus enrichmentHere we describe a manual workflow that takes less than15 minutes to enrich intact viruses from dilute cell culture, viral transport medium (VTM), and wastewater, with an optional step (~10 minutes) to release the virus from beads. This short and simple enrichment approach helps reduce the risk of lower yields and affecting the integrity and infectivity of the virus. One of the key features of Invitrogen™ Dynabeads™ magnetic beads in any enrichment or isolation protocol is the rapid binding kinetics. The(e.g., western blot), or nucleic acid (NA) extraction (e.g., for qRT- PCR). For details on the manual and automated protocols, see Dynabeads Intact Virus Enrichment.Bead-based target enrichment workflowsAproximity of the beads to the targets in the solution translates directly to short incubation times and faster protocols (Figure 1A). Here we utilize the physical properties of viruses (negatively charged) in combination with positively charged Invitrogen™ Dynabeads™ Intact Virus Enrichment beads, which leads toAdd Dynabeadsmagnetic beads to sampleBMix and incubatefor 10 minutesWash bead–targetcomplexAdd release bufferand incubate for 10 minutescharge-based binding without the need for antibody labeling (Figure 1B). This approach can also capture other negatively charged vesicles (e.g., exosomes) or proteins, within 10 minutesPrepare KingFisherplates with Dynabeads magnetic beads, virus sample, and buffersLoad plates intoKingFisher instrument according to script; press “Start”Collect sample (viruson Dynabeads magnetic beads, or released virus)(Figure 2A). Following capture, the viruses can be released from the beads by adding an anion with a stronger relative affinity than that of the virus.This short and easy enrichment approach can be simplified further by using the Thermo Scientific™ KingFisher™ Purification System (Figure 2B). The enriched viruses can be used for functional studies, immunological studies, protein analysisFigure 2. Overview of the bead-based target enrichment workflows.(A) Manual and (B) automated workflows.Outline of viral enrichment and analysisThis study demonstrates the use of the positively charged Dynabeads Intact Virus Enrichment beads to efficiently enrich SARS-CoV-2 from various starting matrices, such as viral transport medium, cell culture medium, or wastewater spiked with virus-like particles or inactivated viruses. The enrichmentShort distanceABRapid kineticsIon exchangeCl–Cl– Cl–Cl–Cl–Cl–Cl–Cl–Cl– Cl– Cl–Cl–Cl– Cl–Cl– Cl–Cl–facilitates downstream analysis by techniques like qRT-PCR and western blot. Additionally, the infectivity of the enriched viruswas assessed by plaque assays. This demonstrates the potential of positively charged beads in facilitating viral enrichment and subsequent analysis in various research applications.Reversible processFigure 1. Binding kinetics and principle of enrichment. (A) The positively charged Dynabeads Intact Virus Enrichment beads are close to the negatively charged virus particles, resulting in rapid binding kinetics and a fast enrichment protocol. Dynabeads Intact Virus Enrichment beads are protected with Cl– ions. Virus particles added to the magnetic beads will displace the Cl– ions and bind to the bead surface. (B) For virus release, an anion with higher relative affinity can be added to displace the viruses and thus release them into the sample. For release, 20 mM triethanolamine with 0.25 M KI or 50 mM citric acid in 50 mM Na phosphate, pH 4, can be used.Viral transport medium50 kDaNStdDir VLPKingFisherManualA Cell culture medium N50 kDaStdDir VLPKingFisherManualBFigure 3. Enrichment of SARS-CoV-2 virus-like particles (VLPs) using Dynabeads Intact Virus Enrichment beads. VLPs were spiked into (A) viral transport medium and (B) cell culture medium and were captured with Dynabeads Intact Virus Enrichment beads using manual and automated protocols. In both, SARS-CoV-2 nucleocapsid protein (N) was detected by western blot.Viral transport medium27.527262524232226.3126.1526.45 26.6125.72MagMAX control Dynabeads272625242322Cell culture medium26.9826.125.56 25.8825.9726.27MagMAX controlDynabeadsWastewater2423.8723.1323.32232222.6322.33 22.3421201918PBSDynabeadsN gene orf1abS geneAverage CtABEnrichment methods and resultsEnrichment of SARS-CoV-2 VLPs from VTM and CCM for protein analysisFor virus enrichment and detection of SARS-CoV-2 nucleocapsid protein N (50 kDa) by WB, VLPs were spiked into VTM or CCM, followed by a 10-minute enrichment using Dynabeads Intact Virus Enrichment beads. The enrichment was performed manually or automated using the KingFisher Flex system (Figure 2).The WB analysis demonstrated similar enrichment efficiency with the manual and automated methods, as determined by the relative intensity of the N protein isolated from both VTM and CCM (Figure 3). This demonstrated that SARS-CoV-2 VLPs can be isolated quickly and efficiently with Dynabeads Intact Virus Enrichment beads.Enrichment of heat-inactivated SARS-CoV-2 virus from VTM, CCM, and wastewater for nucleic acid analysis For enrichment and nucleic acid (NA) detection of SARS-CoV-2 genes (N, orf1ab, and S) by qPCR, heat-inactivated SARS-CoV-2 virus was spiked into VTM, CCM, or wastewater followed by a 10-minute enrichment using Dynabeads Intact Virus Enrichment beads. Extraction of NA was performed using the Applied Biosystems™ MagMAX™ Viral/Pathogen II Nucleic Acid Enrichment Kit (VTM and CCM, Figures 4A and 4B) or MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (wastewater,Figure 4C) after virus enrichment, followed by downstream analysis using the Applied Biosystems™ TaqPath™ COVID-19 Combo Kit.Average CtThe results demonstrated that the qRT-PCR sensitivity with beads-enriched VTM, CCM, and wastewater samples matched the sensitivity with the positive controls (directly extracted for nucleic acids, no bead enrichment; Figure 4)—the Ct values werewithin 2 cycles of the respective controls.Average CtCFigure 4. Enrichment of heat-inactivated SARS-CoV-2 virus. The virus was enriched from (A) VTM, (B) CCM, and (C) wastewater. The N, orf1ab, and S genes of SARS-CoV-2 were detected by qRT-PCR.Enrichment of infectious SARS-CoV-2 from cell culture mediumAverage CtFor enrichment of contagious viruses, SARS-CoV-2 was collected from infected patients and was transferred to VTM. Vero cells were infected with the collected virus for 48 hours, and the viruses produced by the cells were released into the CCM and enriched by either centrifugation, precipitation using Invitrogen™ Intact Virus Precipitation Reagent, or using Dynabeads Intact Virus Enrichment beads. The amount of infectious viral particles produced was determined by counting the number of plaque- forming units on a monolayer of target cells after seeding the monolayer with the isolated virus (Ragon Institute of MGH,MIT, Harvard, USA). The counted plaques for each enrichment method are represented as fold increases compared to no virus enrichment (Figure 5). Both Dynabeads magnetic beads–based and precipitation-based enrichment resulted in higher yieldsof infectious SARS-CoV-2 when compared to enrichment by centrifugation alone (Figure 5B).A30282624222018PBSDynabeadsAdenovirus Influenza A Enterovirus NorovirusFigure 6. Enrichment of adenovirus, influenza A virus, enterovirus, and norovirus from wastewater, and detection by qRT-PCR.Enrichment of other infectious virusesFor enrichment of other contagious viruses, Dynabeads Intact Virus Enrichment beads were added to samples containing influenza virus A, RSV, or Zika virus (ZIKV). The enrichment was assessed by western blot (Figure 7) or virus titers (Figure 8). The presence of influenza A nucleoprotein, RSV fusion protein, and Zika virus NS1 protein in samples enriched with Dynabeads Intact Virus Enrichment beads was confirmed by western blot (Figure 7). Different dilutions of bead-bound virus samples wereSARS-CoV-2 in viral transport mediumB 109Fold increase87654321SARS-CoV-2 cultured in cell culture mediumSARS-CoV-2harvested with DynabeadsEstimation of viral titer by plaque assayprepared for plaque assay analysis. Plates with plaques used for calculation of virus titers are shown in Figure 8. The virus titers were 3.3 x 105 PFU/mL for influenza A virus, 2.6 x 104 PFU/mL for RSV, and 5.0 x 105 PFU/mL for Zika virus.Influenza A virus Zika virus RSV 50 kDa50 kDa 50 kDaStd Isol  –    + Std Isol   –    + Std Isol  –    +  0Centrif Dynabeads PrecipitationFigure 5. Enrichment of infectious SARS-CoV-2 supernatant.(A) Infectious SARS-CoV-2 was cultured in Vero cells and harvested after 48 hours. Viral titers after enrichment were estimated by plaqueassay. (B) Viruses were concentrated by centrifugation, Dynabeads Intact Virus Enrichment beads, or precipitation, and seeded onto a monolayer of cells. The numbers of viruses were estimated by counting plaques (image inset).Enrichment of viruses from wastewater for NA analysis For enrichment and NA detection of other viruses, inactivated adenovirus, influenza A virus, norovirus, and enterovirus were spiked into 10 mL of wastewater. The viruses were enriched within 10 minutes using Dynabeads Intact Virus Enrichment beads, followed by RNA isolation using the MagMAX Microbiome Ultra kit. The results demonstrated that the Dynabeads Intact Virus Enrichment beads can isolate other negatively charged viruses besides SARS-CoV-2, and that qRT-PCR sensitivity is comparable between the enriched samples and the respective PBS controls (Figure 6).Ctrl Ctrl Ctrl Virus dilutions –1 –2 –3 No virus Influenza AvirusRSVZika virusFigure 7. Enrichment of influenza A virus (H1N1), Zika virus, and RSV with Dynabeads Intact Virus Enrichment beads. Influenza A virus, Zika virus, and RSV were spiked into CCM and were captured with Dynabeads Intact Virus Enrichment beads using manual protocols, and analyzed by western blot.Figure 8. Enrichment of influenza A virus, RSV, and Zika virus with Dynabeads Intact Virus Enrichment beads. Viral titers after enrichment were estimated by plaque assays.SummaryWe have described an easy, rapid, and reliable bead-based method to capture a variety of viruses and VLPs by taking advantage of the strong anion-exchange principle. Virus enrichment utilized both manual and automated protocols. Enrichment was performed with Dynabeads Intact Virus Enrichment beads for multiomic analysis (qRT-PCR, western blot, and plaque assay). We demonstrated the successful isolation and release of intact infectious viruses. The automated protocolfor rapid and efficient enrichment of viruses is compatible with the KingFisher Flex, Duo Prime, and Apex systems. Themethods help provide investigators an easy, fast solution for the rapid enrichment of intact viruses for multiomic viral research.Ordering informationDescription Cat. No.Dynabeads Intact Virus Enrichment 10700DIntact Virus Precipitation Reagent 10720DKingFisher Flex Purification System with 96 Deep-Well Head A32681KingFisher 96 Deep-Well Plate, V-bottom, polypropylene (50–1,000 µL) 95040450KingFisher 96 Tip Comb for Deep-Well Magnets 97002534BindIt 4.0 Software (Dynabeads Intact Virus Enrichment-Flex script for download) See 10700DDynaMag-2 Magnet 12321DHulaMixer Sample Mixer 15920D4X Bolt LDS Sample Buffer B000710X Bolt Sample Reducing Agent B0004Bolt 4–12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well NW04120BOXiBlot 2 Gel Transfer Device IB2101iBind Western System SLF1000Goat Anti–Mouse IgG1 Cross-Adsorbed Secondary Antibody, HRP A10551SARS/SARS-CoV-2 Coronavirus Nucleocapsid Monoclonal Antibody MA5-29981 Learn more at For Research Use Only. Not for use in diagnostic procedures. © 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL36169 0624Application note | Dynabeads magnetic beads‌Magnetic bead–based workflowsIsolation of circulating tumor cells using Dynabeads magnetic beadsIntroductionCirculating tumor cells (CTCs) are gaining importance as prognostic markers and for monitoring of treatment response. Because of the low number of CTCs in circulation, highly sensitive methods are necessary to capture and detect down to single cells.Invitrogen™ Dynabeads™ magnetic beads provide an automation-friendly tool for isolation of circulating biomarkers.Generally, large beads are optimal for working on open platforms, while smaller beads are optimal for microfluidics. Positive isolation can be utilized to separate CTCs expressing cancer-specific markers, whereas negative isolation can be utilized to deplete leukocytes from blood samples for marker-independent CTC enrichment, leaving the target cells untouched. Here we evaluate Dynabeads magnetic beads for feasibility in both positive and negative CTC isolation workflows.MethodsPositive isolation of CTCs was evaluated by utilizing Dynabeads magnetic beads coupled to monoclonal antibodies (Abs) targeting epithelial cell adhesion molecule (EpCAM). Contrived samples were prepared at a titration of 1, 3, 5, 7, and 10 epithelial cancer cells spiked into 7.5 mL of whole blood diluted 1:2 in Thermo Scientific™ Phosphate-Buffered Saline (DPBS), Dulbecco’s formula, using a micromanipulator to create five independent samples of varying cell concentrations. Invitrogen™ Dynabeads™ Epithelial Enrich (M-450) magnetic beads* were used to capture CTCs from whole blood samples. The captured cells werethen washed and lysed directly on the Dynabeads magnetic beads using a magnetic stand. For control samples, single cells were lysed directly. The lysates were mixed with Invitrogen™ Dynabeads™ Oligo(dT)25 beads for mRNA isolation and downstream RT-qPCR analysis. The cells were quantified using Applied Biosystems™ TaqMan™ Assay chemistry targeting cytokeratin 19 (CK19). Furthermore, Invitrogen™ Dynabeads™ MyOne™ Epoxy magnetic beads* coupled to anti-EpCAM Abs and Invitrogen™ Dynabeads™ MyOne™ Streptavidin C1 and T1 beads coated with biotinylated anti-EpCAM Abs were compared to Dynabeads Epithelial Enrich magnetic beads at a concentration of 5 cells/sample from four different donors. RT-qPCR with TaqMan Assays was used to analyze expression of CK19 andCD45 from CTCs and white blood cells, respectively, as a measure of cell capture. Figure 1 details the general workflow proposed for positive isolation to capture epithelial cells from whole blood samples.* M-450 beads are 4.5 µm. MyOne beads are 1 µm.TTT TTT Collect sampleCapture epithelial cellsWash and lyse cellsIsolate mRNAAnalyze using RT-qPCRRT-qPCRT T TT T TT T TT T TFigure 1. Positive isolation workflow for detection of CTCs using Dynabeads magnetic beads coupled to anti-EpCAM Abs for epithelial cell capture. Following capture, magnetic beads can be washed using a magnetic stand, and subsequent lysate can be utilized for mRNA isolation to quantify cells with expression markers using RT-qPCR.Negative isolation of CTCs was evaluated by depleting CD45- positive leukocytes collected from blood-derived samples using Dynabeads magnetic beads coupled to anti-CD45 Abs, allowing for CTCs to remain unbound to beads as they are free of this surface marker. Mononuclear cells (MNCs) were isolated from buffy coat and incubated with Invitrogen™ Dynabeads™ CD45 beads (M-450) for leukocyte depletion. The sample containing CTCs was removed from the leukocytes bound tothe beads using a magnetic stand. After magnetic separation, the cells remaining in the supernatant were stained with PE- labeled anti-CD45 and analyzed by flow cytometry for percent depletion. Dynabeads CD45 beads were compared to different surface-activated MyOne beads as well as to streptavidin beads coated with the same anti-CD45 antibody. Figure 2 details the general workflow proposed for negative isolation to capture and remove leukocytes using Dynabeads CD45 beads.Collect sample Capture leukocytes Remove cells Analyze cellsFigure 2. Negative isolation workflow for the capture and removal of leukocytes bound to Dynabeads CD45 beads. In this study, flow cytometry was utilized to evaluate bead performance in negative isolation workflows.Results and discussionThe Ct values for CK19 expression from cells captured using the positive isolation workflow described above were within the range determined by the controls, indicating highly sensitive capture down to one single cell when comparing 1:2-diluted whole blood samples containing 1, 3, 5, 7, or 10 cells to the control samples(Figure 3).Real-time PCR results were comparable across all Dynabeads platforms, indicating similar efficiency in cell capture (Figure 4A). However, the amount of nonspecific binding of leukocytes was lower for Dynabeads MyOne Streptavidin C1 and T1 beads(~10x lower; ΔCt = 3.4), compared to Dynabeads Epithelial Enrich beads (Figure 4B).Flow cytometry histograms confirmed cell count reduction post-depletion using Dynabeads CD45 beads (Figure 5).Figure 6 shows a comparison of depletion between Dynabeads CD45 beads and Dynabeads MyOne Epoxy beads coupledto anti-CD45 Abs. A higher number of beads per cell required for Dynabeads MyOne beads is due to the smaller bead size compared to that of Dynabeads CD45 beads. However, note thetotal bead mass was ~10x lower with the Dynabeads MyOne magnetic beads. Dynabeads MyOne beads with different surface activations and the corresponding streptavidin beads coated with the same anti-CD45 antibody revealed different depletion efficiencies (Figure 7).343332 Ct value3130 29 2827 0 2 4 6 8 10Number of epithelial cellsFigure 3. Ct values obtained for different number of cells. Single cells (1, 3, 5, 7, and 10 cells) were spiked into and captured from 7.5 mL of1:2-diluted whole blood (red) or lysed directly (controls; blue).A 35Ct value3025Donor 1 Donor 2 Donor 3 Donor 4Dynabeads magnetic beadsB 35Ct value3025Donor 1 Donor 2 Donor 3 Donor 4Dynabeads magnetic beadsMyOne Epoxy MyOne Streptavidin C1 MyOne Streptavidin T1 Epithelial EnrichMyOne EpoxyMyOne Streptavidin C1 MyOne Streptavidin T1 Epithelial EnrichFigure 4. Comparison of RT-qPCR results from positive CTC isolation workflows. Five cells were spiked into blood from four donors and captured using four types of Dynabeads magnetic beads coupled to anti-EpCAM Abs. (A) CK19 and (B) CD45 expression levels were analyzed.A400350300Count250200150100500102 103 104 105CD45 PE-AB400350300Count250200150100500102 103 104 105CD45 PE-AFigure 5. Fluorescence histograms for CD45-positive MNCs and scatter plots analyzed using flow cytometry (A) before and (B) after depletion using Dynabeads CD45 magnetic beads.Epoxy-activated beads showed the highest depletion efficiency while carboxyl acid–activated beads demonstrated the least efficiency. However, different MyOne Streptavidin beads coated with anti-CD45 Abs showed only minor differences.Different size beads exhibited different binding properties. Small beads have been shown to be beneficial for the depletion of cells with low expression of surface markers. Dynabeads CD45 magnetic beads depleted only ~80% of CD45-low granulocytes (Figure 8). Cell depletion improved when Dynabeads CD15 magnetic beads were added, which directly targeted granulocytes. In contrast, applying anti-CD45 coupled120100Depletion (%)8060     40     20     020 40 20 + 20 125CD45 CD45 + CD15 CD45MyOne beadsDynabeads M-450 beads per cell DynabeadsDynabeads MyOne beads achieved similar depletion efficiencyTotal Lymphocytes Monocytes Granulocytesper cellas that of the combination of Dynabeads CD45 and CD15 magnetic beads.120                                                                                                        100Depletion (%)806040200CD45Figure 8. White blood cell depletion efficiency after RBC lysis of Dynabeads CD45 (M-450), CD15 (M-450) beads, and MyOne Epoxy beads coupled with anti-CD45 Abs.ConclusionsThe findings obtained from this study revealed that Dynabeads magnetic beads can isolate CTCs with high specificity and sensitivity for both positive and negative isolation techniques. Dynabeads magnetic beads can be coupled to antibodies targeting cancer-specific markers, such as EpCAM, for direct cell capture, or to anti-CD45 Abs for leukocyte depletion for CTC enrichment, demonstrating feasibility for both positive and negative isolations. Flow cytometry results using negative isolation workflows and Dynabeads magnetic beads revealedbeadsBeads per cellDynabeads MyOne magnetic beads are more efficient atFigure 6. Cell depletion efficiency of Dynabeads CD45 beads, and Dynabeads MyOne Epoxy beads coupled to anti-CD45 Abs, determined by flow cytometry.120                                                                                               100                                                                                               Depletion (%)80      60      40      20      capturing cells with low expression of surface markers (e.g., granulocytes expressing low CD45) compared to Dynabeads CD45 magnetic beads. However, this is not relevant when MNCs are used as the starting material. Although Dynabeads MyOne magnetic beads do require a higher number of beads, lesstotal bead mass is required to achieve efficient cell capture or depletion in comparison to that of 4.5 µm magnetic beads.AuthorsFrauke Henjes, Elisabeth Breivold, Marie Bosnes, Hannah Lindstrom, Thermo Fisher Scientific,Oslo, Norway0DynabeadsCD45beadsCarboxylic acid– activatedEpoxy Dynabeads MyOne beadsTosyl- activatedC1 T1Dynabeads MyOne StreptavidinAcknowledgmentThank you to Thermo Fisher Scientific research and development team for their dedication, professional experience, andFigure 7. Comparison of cell depletion efficiency using differentsurface-activated Dynabeads MyOne beads, Dynabeads CD45 beads, and MyOne Streptavidin beads. C1 = carboxylic acid– activated; T1: tosyl-activated.commitment to this study. Learn more at For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL35927 1223APPLICATION NOTE MagMAX Cell-Free DNA Isolation Kit‌Extraction and analysis of circulating cell-free DNA from plasma samples for lung cancer researchAbstractWe report a method for the extraction of circulating cell-free DNA (cfDNA) from plasma samples using theApplied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit followed by digital PCR and next-generation sequencing analysis. Extraction and analysis of cfDNA was successfully performed from the plasma of 3 donors in good healthand 11 donors with lung cancer. The results show that the MagMAX Cell-Free DNA Isolation Kit offers efficient recovery of high-quality cfDNA for downstream research applications and enables time savings compared to traditional methods.IntroductionDevelopment of drugs targeting driver genes involved in lung cancer progression has become essential for the advancement of treatment options. In addition, thetolerance acquisition mechanisms of cells to these targeted therapies has been clarified, leading to the development of second- and third-generation drugs to allow post-tolerance therapy strategies. Companion diagnostics to help confirm tolerance acquisition mechanisms after primary treatment is indispensable for these new generations of drugs.Since it is often difficult to conduct an invasive second biopsy during or after treatment, there is considerable interest in developing less-invasive genetic tests that utilize peripheral blood. It is well known that the peripheral blood of cancer patients contains tissue-derived cfDNA [1]. As genetic abnormalities have been analyzed using cfDNA formany cancer types such as lung cancer, breast cancer, pancreatic cancer, colon cancer, and prostate cancer, the interest in developing minimally invasive genetic tests has increased.Lung cancer is a leading cause of death worldwide, with the majority of cases attributed to non-small cell lung carcinoma [2]. Mutation of the epidermal growth factor receptor gene (EGFR) is frequently observed in non-small cell lung carcinoma. For lung cancers positive in this mutation, first-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) are applied, which often have a remarkable effect, but eventually develop tolerance [3]. The T790M mutation of EGFR is recognized in approximately half of drug-resistant tumors, and third-generation EGFR-TKIs are effective against lung cancers with this mutation.Before treatment with targeted therapies, it is essential to confirm the T790M mutation using companion diagnostics.As previously described, it is often difficult to perform a second biopsy of the tumor tissue, so genetic testingusing cfDNA from peripheral blood is of interest to many researchers studying practical methods to replace a tissue biopsy.Genetic testing using cfDNA has significant challenges that are not typically associated with traditional testing using tumor tissues. First, the test must be compatible with a very small amount of nucleic acid, since cfDNA can be collected from only a few milliliters of peripheral blood. Second, the test requires a highly sensitive method that can detect a tumor-derived cfDNA mutation existing at low frequency, since peripheral blood also contains circulating DNA derived from normal cells. These challenges canbe overcome with highly sensitive detection technologies such as digital PCR as well as simple methods to efficiently extract cfDNA from peripheral blood. Here we report the results of an evaluation of the MagMAX Cell-Free DNA Isolation Kit by Prof. Kazuto Nishio and Dr. Kazuko Sakai, Department of Genome Biology, KINDAI University, Faculty of Medicine, Osaka, Japan.Materials and methodsAfter donor consent and approval by the ethics committee of the university, peripheral venous blood from 3 donors in good health and 11 donors with lung cancer were collected using EDTA blood collection tubes, from which the plasma was separated by centrifugation.Extraction of cfDNA from approximately 1 mL plasma was performed as described in the MagMAX Cell-Free DNA Isolation Kit user guide. A Bioanalyzer™ instrument (Agilent) was used to confirm the size of extracted DNA. The yield of cfDNA was calculated by quantifying the number of copies of the RNaseP gene using Applied Biosystems™ TaqMan™ Copy Number Assays.ResultsFluorescenceUsing plasma samples from healthy donors, an average of 581 copies (maximum 716 copies, minimum 404 copies) of cfDNA per 1 mL of plasma was extracted (Table 1). Bioanalyzer instrument results showed that only DNA near 170 bases was present after extraction, which is normally considered to be derived from cfDNA,and high molecular weight DNA was not found (Figure 1). Since macromolecular DNA is primarily derived from the mononuclear cells mixed in the plasma, it becomes a hindrance in subsequent experiments and analysis. Using plasma samples from donors with lung cancer, an average of 4,127 copies (maximum 10,477 copies, minimum 974 copies) of cfDNA was extracted from 0.5 mL to 1.9 mL of plasma (Table 2). Extracted cfDNA was also examined for the EGFR T790M mutation using digital PCR and amplicon sequencing using the Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2. High-quality somatic mutation and sequencing analysis results were obtained (data not shown).Table 1. cfDNA yield from plasma samples of healthy donors.Table 2. cfDNA yield from plasma samples of donors with lung cancer.DNA size (bp) DNA size (bp) DNA size (bp) Figure 1. Bioanalyzer instrument results for cfDNA extracted from the plasma samples of healthy donors.DiscussionIn this study, cfDNA was extracted from the plasma samples of multiple donors using the MagMAX Cell-Free DNA Isolation Kit. The results indicate that the cfDNA yield per mL of plasma from donors with lung cancer is approximately 6 times higher compared to the cfDNA yield per mL of plasma from healthy donors. The yield of cfDNA obtained is comparable to traditional extraction methods, and is sufficient to analyze by digital PCR, targeted next-generation sequencing, and other genetic analysis methods.It is important to use cfDNA of consistent quality before proceeding with genetic analysis. Results from the Bioanalyzer instrument showed that only DNA around 170 bp was found, and high molecular weight DNA was not detected. Digital PCR and amplicon sequencing werealso readily performed with the extracted cfDNA. Together, these results suggest that high-quality cfDNA was obtained from the kit.As the development of high-throughput technology has progressed, advancements in the ease of use and sample- processing capability of nucleic acid extraction methods are in greater demand. The MagMAX Cell-Free DNA Isolation Kit uses a magnetic bead–based DNA extraction method, which is adaptable to automation. Proteinaseand heat treatment are unnecessary with this kit, enabling all processes to be conducted at room temperature.* Compared to traditional cfDNA extraction methods, there is also a significant reduction in processing and hands-on time (Figure 2). The genome biology class at the KINDAI University School of Medicine extracts cfDNA from as many as 1,000 samples annually. For large numbers of samples, utilizing the MagMAX Cell-Free DNA Isolation Kit instead of traditional methods can help increase workflow efficiencies and speed time to results.MagMAX Cell-Free DNA Isolation KitManual method: ~40 min at room temperaturePellet beads and remove supernatantResuspend beads and transfer to microfuge tubeWash 1x with wash solution, wash 2x with 80%ethanol, dry 3–5 minElute with 50 µL elutionsolution~40 minutesTraditional productManual method: 2–3 hours, heating requiredLyse and bind                                  Vacuum                 Wash 3xCentrifugeElute12 samples60˚C4˚C56˚C 2–3 hoursFigure 2. MagMAX Cell-Free DNA Isolation Kit protocol compared to a traditional column-based protocol.* Applicable when EDTA tubes are used. When Streck tubes are used, a proteinase digestion is recommended. See the product manual for details.ReferencesFind out more at thermofisher.com/cfdnaisolationFor Research Use Only. Not for use in diagnostic procedures. © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Bioanalyzer is a trademark of Agilent Technologies Inc. COL31084 0516APPLICATION NOTE MagMAX Cell-Free DNA Isolation Kit‌A complete next-generation sequencing workflow for circulating cell-free DNA isolation and analysisCirculating cell-free DNA (cfDNA) has been shown to have potential as a noninvasive substrate for the detection and monitoring of tumor cells. As circulating tumor DNA is often present at low frequencies within cfDNA, targeted sequencing is an optimal tool for mutation detection. To support advancement of cfDNA research, we demonstrate a complete workflow providing: (1) isolation of cfDNA fromplasma using the Applied Biosystems™ MagMAX™ Cell-Free DNA Isolation Kit either manually with a magnetic stand or automatically on the Thermo Scientific™ KingFisher™ Flex or KingFisher™ Duo Prime Magnetic Particle Processor, followed by (2) molecular characterization of isolated cfDNA using the multiplexing capabilities of Ion AmpliSeq™ technology and the Ion PGM™ System.cfDNA isolated with the MagMAX Cell-Free DNA Isolation Kit and amplified with the Ion AmpliSeq™ Cancer Hotspot Panel v2 demonstrates reproducible amplicon representation and variant detection across 50 genes of interest, covering 2,800 COSMIC mutations. The performance is comparable whether cfDNA is isolated with the MagMAX Cell-Free DNA Isolation Kit or a column-based protocol. Importantly, performanceis also comparable when using less than half the volume of plasma with the MagMAX Cell-Free DNA Isolation Kit than with column-based protocols. Through saturation studies and subsampling, the limit of detection of hotspots in cfDNA on the Ion PGM System is determined to be below 1%.This robust workflow for cfDNA analysis through targeted sequencing combines simple sample preparation with the ease of Ion AmpliSeq technology and the rapid turnaround time of the Ion PGM System.cfDNA has been shown to have potential as a noninvasive substrate for the detection and monitoring of tumor cells in published literature [1, 2]. As circulating tumor DNA is often present at low frequencies within cfDNA, targetedsequencing is an optimal tool for mutation detection. cfDNA in plasma is widely used for basic and clinical research, including oncology studies. However, some commercially available cfDNA isolation kits have lengthy protocols, use many reagents, and often require heating for Proteinase K treatment and DNA elution, which can make the process difficult to implement.Advances in next-generation sequencing (NGS) technology now enable the use of cfDNA as a biomarker for liquid biopsy research applications. Successful use of cfDNAas an oncology research biomarker requires an easily implemented protocol to access the template and efficient DNA extraction from large volumes of plasma (1–10 mL) owing to the low concentration of cfDNA (10–1,000 copies/ mL) in blood. The ability to concentrate and analyze the limited cfDNA contained in these large plasma volumes with technologies such as NGS enables researchers to obtain more information more quickly than with established methods. The accessibility of blood samples suggests that cfDNA extraction and analysis could be beneficial in cancerresearch for potential detection and monitoring of tumor cell progression in the future.Materials and methods4 mL and 10 mL plasma were prepared from each sample using the MagMAX Cell-Free DNA Isolation Kit and the KingFisher Flex Magnetic Particle Processor with 24 Deep- Well Head. Quantitation was performed with the Agilent™ High Sensitivity DNA Kit to assess the cfDNA fraction and the Invitrogen™ Qubit™ dsDNA HS (High Sensitivity) Assay Kit to quantify total DNA yield.To verify compatibility with NGS analysis, 4 non-small cell lung cancer (NSCLC) cfDNA samples were extracted from archived plasma using either the MagMAX Cell-Free DNA Isolation Kit or a commercially available column-based kit (kit Q). cfDNA was isolated from 1 mL of each plasma sample using the published protocol for kit Q. For extraction using the MagMAX Cell-Free DNA Isolation Kit, cfDNA was isolated from 1 mL for two of the plasma samples, while the sample input was substantially less for the other two plasma samples (0.580 mL and 0.381 mL).Libraries were constructed using the Ion AmpliSeq™ Library Kit 2.0 and the Ion AmpliSeq Cancer Hotspot Panel v2 using 20 cycles of PCR. Template preparation was performedon the Ion Chef™ System followed by sequencing on the Ion PGM System. Variant detection was performed by the<1 hour3.5 hours10.5 hours (30 minutes hands-on time)4.5 hours (excluding initialization)Torrent Variant Caller plugin on Torrent Suite™ Software or Ion Reporter™ Software.ResultsFaster processing of samples can be achieved with automated isolation using the MagMAX Cell-Free DNA Isolation Kit on the KingFisher Flex or KingFisher Duo Prime system. Alternatively, the samples can be processed manually with a magnetic stand. This is followed by atargeted sequencing approach in which library and template are prepared from the enriched cfDNA and sequenced on the Ion PGM System (Figure 1).MagMAX kit cfDNA isolationIon AmpliSeq library preparationIon Chef System template preparationIon PGM System sequencingTorrent Suite Software analysisFigure 1. A scalable and reproducible workflow to successfully analyze cfDNA from large volumes of plasma via NGS.Circulating cfDNA is highly fragmented, with the major fraction around 170 bp. To examine DNA size-dependent recovery, 50 µL of 50 bp DNA ladder was spiked into4 mL of commercially available plasma that was depleted of endogenous cfDNA. The MagMAX Cell-Free DNA Isolation Kit was then used to purify the DNA ladder. Following analysis on the Agilent™ 2100 Bioanalyzer™ system, approximately 100% of cfDNA ranging from 100 bp to750 bp was recovered, while less-efficient elution of DNA≥800 bp was observed (Figure 2). This efficient recovery of double-stranded DNA (dsDNA) <800 bp yields eluted DNA samples specifically enriched for cfDNA.340 bp170 bpTo examine reproducibility, 4 plasma samples were processed in duplicate using the MagMAX Cell-Free DNA Isolation Kit. The extracted DNA was analyzed with a 2% Invitrogen™ E-Gel™ EX Agarose Gel (Figure 3). Each replicate set showed a similar yield of cfDNA with the major fraction clearly visible at ~170 bp.50 bp DNA ladder input DNA ladder recovered Figure 2. Efficiency of short DNA recovery. 60 µL of MagMAX™ Cell- Free DNA Magnetic Beads was added to 4 mL of cfDNA-depleted plasma that was spiked with 50 µL of 50 bp DNA ladder (2 ng/µL). Bound DNA was washed and eluted in 50 µL elution solution. To assess recovery efficiency of the MagMAX Cell-Free DNA Isolation Kit, 1 µL of ladder and 1 µL of the extracted DNA sample were run on the Agilent 2100 Bioanalyzer system using the High Sensitivity DNA Chip.Figure 3. Reproducibility of cfDNA recovery. Plasma samples from 4 donors were processed in duplicate using the MagMAX Cell-Free DNA Isolation Kit. cfDNA was extracted from 4 mL plasma and eluted in 50 µL elution solution. 20 µL of eluted DNA was run on a 2% E-Gel EX Agarose Gel for analysis.MagMAX kit Protein carryover (µg) Protein contamination in DNA extracted with the MagMAX Cell-Free DNA Isolation Kit and a commercially available column-based kit (kit Q) was quantified with the Invitrogen™ Qubit™ Protein Assay Kit. The samples processed with the MagMAX Cell-Free DNA Isolation Kit show considerably less protein carryover as compared to the competitor kit even though the MagMAX kit protocol does not utilize Proteinase K (Figure 4). The protocol for kit Q starts with a 30-minute Proteinase K digestion at 60°C.Figure 4. Protein carryover after DNA isolation. Protein was quantified using the Qubit Protein Assay Kit following DNA isolation with the MagMAX Cell-Free DNA Isolation Kit or kit Q, a column-based kit.Some high molecular weight cellular DNA may be released from white blood cells after blood draw and during plasma separation. cfDNA is highly fragmented and co-migrates with ribosomal RNA with a major peak at ~170 bp. By design, the MagMAX Cell-Free DNA Isolation Kit efficiently recovers only DNA shorter than 800 bp (Figure 2), and thus has the potential to enrich for cfDNA. This was confirmed by comparing cfDNA enrichment with the MagMAX Cell-Free DNA Isolation Kit to enrichment with kit Q using various plasma samples (Figure 5). Compared to kit Q, the MagMAX Cell-Free DNA Isolation Kit demonstrates equivalent cfDNA (<700 bp fraction) yield but lower cellular DNA (>700 bp fraction) yield. Thus, the cfDNA fraction as a percentage of total extracted DNA was higher (i.e., cfDNA was enriched) for the MagMAX Cell-Free DNA Isolation Kit.The sample volume processed and DNA recovered are highly scalable using the MagMAX Cell-Free DNA Isolation Kit. dsDNA of 120 bp and 170 bp in length were spiked intoplasma samples at varying input amounts ranging from 10 ng to 200 ng. Consistent, efficient recovery of both DNA spikes is observed over the input range (Figure 6A). To demonstrate sample volume scalability, cfDNA from 4 mL and 10 mL plasma were isolated from the same sample. As expected, DNA yield from 10 mL plasma was ~2.5x greater than that from the 4 mL plasma sample (Figure 6B and C), while total protein carryover only marginally increased (Figure 6D).Processing up to 5 mL of plasma can be automated with the KingFisher Flex (24 samples per run) or the KingFisher Duo Prime (6 samples per run) system. Enriched cfDNA samples are ready in about 40 minutes after reagents and plasma samples are loaded into the plates. To demonstrate the performance of both systems, 4 mL per sample was processed from 4 plasma samples using the MagMAX Cell- Free DNA Isolation Kit. The eluted DNA was loaded onto a High Sensitivity DNA Chip and run on the Agilent Bioanalyzer 2100 system. The yield and size profile are approximately equivalent for all 4 samples, indicating similar recovery performance (Figure 7).Sample 1Sample 2Sample 3Sample 4Figure 5. Enrichment of cfDNA following extraction from plasma samples. Cell-free plasma was separated from 4 normal blood samples by centrifugation for 20 minutes at 2,000 x g, then for 30 minutes at 6,000 x g. DNA was extracted from 4 mL plasma using either the MagMAX Cell-Free DNA Isolation Kit (red trace) or kit Q (blue trace). The eluted DNA was loaded onto a High Sensitivity DNA Chip and run on the Agilent Bioanalyzer 2100 system.DNA: 120 bp DNA: 170 bp dsDNA input (ng)Plasma IDcfDNA100–275 bp,Bioanalyzer system Protein, Qubit assayPlasma IDTotal DNA, qPCR Plasma IDTotal human DNA yield (ng)dsDNA recovered (ng) Protein carryover (µg)cfDNA, 100–275 bp (ng)Figure 6. Scalability of cfDNA isolation. (A) 120 bp and 170 bp dsDNA were spiked into plasma samples at varying input amounts and recovered using the MagMAX Cell-Free DNA Isolation Kit. (B) cfDNA from 4 mL and 10 mL plasma quantified using the Agilent Bioanalyzer 2100 system. (C) cfDNA from 4 mL and 10 mL plasma quantified by qPCR. (D) Total protein carryover as measured using the Qubit Protein Assay Kit.Sample 1Sample 2Sample 3Sample 4Figure 7. Automated cfDNA isolation using the KingFisher systems. Overlapping traces for cfDNA isolated with the KingFisher Flex (red) and KingFisher Duo Prime (blue) systems are shown.Four NSCLC cfDNA samples were isolated using the MagMAX Cell-Free DNA Isolation Kit or kit Q. For kit Q, each sample was isolated from 1 mL of plasma. For the MagMAX Cell-Free DNA Isolation Kit, samples 1 and 2 were isolated from 1 mL of plasma, while sample 3 was isolated from 0.580 mL of plasma and sample 4 was isolated from0.381 mL of plasma. Ion AmpliSeq™ libraries were prepared with the Ion AmpliSeq Cancer Hotspot Panel v2, template was prepared on the Ion Chef System, and sequencing was performed on the Ion PGM System. The percent of sequencing reads that were on target were comparable between the two isolation methods, including when a lower volume of plasma was used for the MagMAX Cell-Free DNA Isolation Kit (Figure 8). The uniformity of the amplicons was comparable for all 4 samples.120100806040200on target (%)uniformity (%)Sample 1Sample 2Sample 3Sample 4MagMAXkit QMagMAXkit QMagMAXkit QMagMAXkit QFigure 8. Amplicon performance of NSCLC samples. Samples 1–4 were isolated using the MagMAX Cell-Free DNA Isolation Kit or kit Q and sequenced on the Ion PGM System. The percent of on-target sequencing reads and uniformity are shown for both isolation methods.Triplicate libraries were prepared using the Ion AmpliSeq Cancer Hotspot Panel v2 from NSCLC cfDNA isolated using the MagMAX Cell-Free DNA Isolation Kit or kit Q. Template preparation and sequencing was performed independently for each library. The insert length versus normalized amplicon coverage as reported by the Coverage Analysis plugin provided in Torrent Suite Software shows a high degree of reproducibility between the two cfDNA isolation protocols (Figure 9A). The percent GC content present in each amplicon versus the normalized amplicon coverage also shows reproducible results for the two cfDNA isolation protocols (Figure 9B).ABFigure 9. Reproducibility of amplicon coverage in NSCLC samples. Triplicate libraries were independently prepared and sequenced from cfDNA isolated usingthe MagMAX Cell-Free DNA Isolation Kit or kit Q. (A) Insert length plotted againstnormalized amplicon coverage. (B) Percent GC content in each amplicon plotted against normalized amplicon coverage.There was good agreement in allelic frequencies using the Torrent Variant Caller plugin provided in Torrent SuiteSoftware. Sample 1 had triplicate cfDNA libraries prepared from the MagMAX Cell-Free DNA Isolation Kit and triplicate cfDNA libraries prepared from kit Q (Table 1). All six libraries showed comparable variant detection results. Sample 3 had 0.381 mL of plasma used for the MagMAX Cell-Free DNA Isolation Kit versus 1 mL used for kit Q and showed comparable variant detection results.MagMAXTable 1. Allelic frequencies for hotspots in NSCLC samples.Sample 3We have developed a workflow with a set of reagents and tools for recovery and analysis of cfDNA from cell-free samples such as plasma or serum. The complete workflow includes automated isolation of cfDNA and sensitive, reproducible downstream analysis via targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2.The Ion AmpliSeq Cancer Hotspot Panel v2 has reproducible amplicon performance and variants detected with cfDNA. The performance of Ion AmpliSeq technology is comparable when the cfDNA has been isolated with the MagMAX Cell- Free DNA Isolation Kit protocol or a column-based method, even when there is less than half the volume of plasmaused with the MagMAX kit protocol than with the column- based method. This robust workflow for cfDNA isolation and targeted sequencing combines simple sample preparation with the ease of Ion AmpliSeq technology and the rapid turnaround time of the Ion PGM System.ReferencesOrdering informationMagMAX Cell-Free DNA Isolation Kit A29319KingFisher Duo Prime Magnetic Particle Processor 5400110KingFisher Flex Magnetic Particle Processor with 96 Deep-Well Head (for plasma volumes less than 1 mL)KingFisher Flex Magnetic Particle Processor with 24 Deep-Well Head (for plasma volumes less than 5 mL)54006305400640Qubit dsDNA HS Assay Kit Q32854Ion AmpliSeq Library Kit 2.0 4480442Ion AmpliSeq Cancer Hotspot Panel v2 4475346Find out more at thermofisher.com/cfdnaisolationFor Research Use Only. Not for use in diagnostic procedures. © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agilent and Bioanalyzer is a trademark of Agilent Technologies, Inc. CO210852 1015Application note | MagMAX DNA Multi-Sample Ultra 2.0 Kit‌Sample prepGenomic DNA extraction from bone marrow aspirates and peripheral blood mononuclear cellsIntroduction and backgroundBone marrow aspirates (BMAs) and peripheral blood mononuclear cells (PBMCs) are commonly used sample types that require reliable sample preparation technology for the research of various diseases. The nucleic acid extracted from BMAs and PBMCs can be used for multiple applications, such as next-generation sequencing (NGS) using the Ion Torrent™ Oncomine™ Myeloid Research Assay, microarray analysis using Applied Biosystems™ CytoScan™ and CytoScan™ HD arrays, real-time PCR using SNP genotyping assays, and many more.The Applied Biosystems™ MagMAX™ DNA Multi-Sample Ultra 2.0 Kit uses magnetic beads that are compatible withsemiautomated or automated genomic DNA (gDNA) extractions from whole blood, saliva, buffy coat, and buccal swabs. Here we evaluate a workflow using the MagMAX DNA Multi-Sample Ultra 2.0 Kit to extract gDNA from BMAs and PBMCs on the Thermo Scientific™ KingFisher™ Duo Prime Purification System. Applied Biosystems™ MagMAX™ Cell and Tissue DNA Extraction Buffer is used for initial processing of samples. High-quality gDNA is produced using this workflow, demonstrated by DNA integrity analysis and real-time PCR performance.Experimental proceduresTwo studies were performed using the MagMAX DNAMulti-Sample Ultra 2.0 Kit to evaluate gDNA extracted from BMAs and PBMCs.In the first study, three BMA samples from different donors were processed in duplicate extractions using the sample preparation workflow shown in Figure 1. Sample volume of 200 µL was centrifuged at 200 x g for 10 minutes in a 1.5 mL Eppendorf™ tube to pellet cells from the BMA sample. The supernatant was removed while being careful not to disturb the pellet formedat the bottom of the tube. The pellet was then resuspended in 400 µL of MagMAX Cell and Tissue DNA Extraction Buffer (included with the MagMAX DNA Multi-Sample Ultra 2.0 Kit in Cat. No. A45721). From this point, the protocol for isolation of DNA from 200–400 µL of whole blood on the KingFisher Duo Prime instrument (Pub. No. MAN0017325) was used with the resuspended cell pellet, instead of a whole-blood sample.In the second study, three PBMC samples from different donors were processed in duplicate extractions using the workflowSample collectionTransfer sample to 1.5 mL tubeCentrifugeRemove supernatantResuspend in MagMAX Cell and Tissue Extraction BufferPerform extractionin Figure 1. Sample input volume of 1 x 10⁶–2 x 10⁶ cells wastransferred to a 1.5 mL Eppendorf tube and centrifuged at 500–800 x g for 10 minutes to pellet the cells. The supernatantwas removed while being careful not to disturb the pellet formed at the bottom of the tube. The pellet was then resuspendedin 400 µL of MagMAX Cell and Tissue DNA Extraction Buffer. From this point, the protocol for isolation of DNA from cultured cells on the KingFisher Duo Prime instrument was followed (Pub. No. MAN0018808).For both studies, extracted double-stranded DNA (dsDNA) was quantified with the Invitrogen™ Qubit™ 1X dsDNA Broad Range (BR) Assay Kit. DNA integrity and size in base pairs (bp) were analyzed on the Agilent™ 4200 TapeStation™ System with a Genomic DNA ScreenTape™ device. To assess DNA qualityusing qPCR, Applied Biosystems™ TaqMan™ Assays (assay IDs: Hs02758991 and Hs03023880) with TaqMan™ Universal Master Mix II, no UNG, were used to detect GAPDH and ACTB gDNA targets on the Applied Biosystems™ ViiA™ 7 Real-Time PCR System, 384-well format. Peripheral blood mononuclear cells (PBMCs)1 x 10⁶–2 x 10⁶ PBMCs 500–800 x g, 10 min RemovesupernatantResuspend in 400 µLMagMAX Cell and Tissue DNA Extraction BufferFollow the cultured cells protocol for the MagMAX DNA Multi-Sample UltraBone marrow aspirates (BMAs)200 µL BMAs200 x g, 10 minResuspend in Follow the whole-bloodprotocol for the MagMAXRemove 400 µL DNA Multi-Sample Ultra supernatant MagMAX Cell 2.0 Kit on a KingFisherand Tissue DNA instrument (400 µLExtraction Buffer sample input)2.0 Kit on a KingFisher instrument (400 µL sample input)Figure 1. Workflows for processing BMAs and PBMCs for gDNA extraction using the MagMAX DNA Multi-Sample Ultra 2.0 Kit and KingFisher instrument.MagMAX DNA Multi-Sample Ultra Kit  thermofisher.com/magmaxultraResults and discussionYields measured with the Qubit 1X dsDNA BR Assay Kit indicated sample-to-sample variability from the DNA recovered from both BMAs and PBMCs (Figures 2A and 2B). Extractions from BMAs resulted in yields as low as 4.54 ng/μL (sample 2) and as high as52.40 ng/μL (sample 3). Yields from PBMCs were consistently high across all three samples at greater than 80 ng/µL.DNA integrity numbers (DINs) for BMAs were greater than 7.0 with strong gDNA peaks observed in theelectropherogram traces across all three samples (Figure 3). For PBMCs, DINs were greater than 9.0 with strong gDNA peaks observed across all samples. qPCR analysis onApplied Biosystems™ ViiA™ 7 Real-Time PCR Software indicated ACTB and GAPDH gDNA targets had strong amplification across all three samples for both sample types (Figure 4).Note: During preparation of sample plates, a white precipitate formed when the samples resuspended in MagMAX Cell and Tissue DNA Extraction Buffer were added to the enhancer solution within a 96 deep-well sample plate. The precipitate dissolved during the proteinase K digestion at high temperature and did not impact DNA recovery.A BConcentration (ng/µL)60                                                                                                                                                                        7.244.5452.40504030201001 2 3140Concentration (ng/µL)120100806040200117.501 2 3Sample SampleFigure 2. Yields of gDNA for three samples. (A) BMA and (B) PBMC samples were processed with the MagMAX DNA Multi-Sample Ultra 2.0 Kit on the KingFisher Duo Prime instrument. Samples were prepared for extraction with the MagMAX Cell and Tissue DNA Extraction Buffer. Both sample extraction methods were evaluated for dsDNA concentration with the Qubit 1X dsDNA BR Assay Kit.ASample 1Sample 2Sample 3BSample 1Sample 2Sample 3Figure 3. DNA integrity and size data. (A) BMA and (B) PBMC samples were processed using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on the KingFisher Duo Prime instrument. Gel representations (left) from the 4200 TapeStation System display DIN results. Electropherogram traces (right) show the integrity and size in bp of recovered DNA.MagMAX DNA Multi-Sample Ultra Kit  thermofisher.com/magmaxultra22.95 22.92                      20.05 20.01                                 A 24.0                                                 23.66   23.72                           23.022.0Ct21.020.019.018.017.0ACTB GAPDHB 20.0                                     19.91                                                      19.6619.49           19.4219.06           18.85                                 19.5Ct19.018.518.017.5ACTB GAPDH1 2 3Sample17.01 2 3SampleFigure 4. qPCR data from processed samples. (A) BMA and (B) PBMC samples were processed with the MagMAX DNA Multi-Sample Ultra 2.0 Kit on the KingFisher Duo Prime instrument. ACTB and GAPDH were amplified from gDNA using TaqMan Assays on the ViiA 7 Real-Time PCR System.Data are from ViiA 7 Real-Time PCR Software.ConclusionsUse of the MagMAX DNA Multi-Sample Ultra 2.0 Kit on a KingFisher instrument can provide a streamlined approach for purification of gDNA from BMA and PBMC samples. This approach expands the type of samples that can be used with the kit and KingFisher platform. The yield and quality of gDNA extracted from BMA and PBMC samples show that the suggested purification workflow is suitable for obtaining high-quality gDNA for various research applications.AuthorsMichelle Leija, Lillie Manley, Thilanka Jayaweera, and Anthony Pedroza, Thermo Fisher ScientificLearn more at thermofisher.com/magmaxultra  For Research Use Only. Not for use in diagnostic procedures. © 2022, 2024 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Eppendorf is a trademark of Eppendorf AG. Agilent, TapeStation, and ScreenTape are trademarks of Agilent Technologies, Inc. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. APN-8148723 0724Application note | AllType and AllType FASTplex NGS assays‌Clinical researchVersatile solutions for human leukocyte antigen testing with peripheral bloodIntroductionThe human leukocyte antigen (HLA) genes are located in the major histocompatibility complex and are considered the most polymorphic genetic system in humans, with more than 35 thousand alleles described and high levels of diversity inside and between human populations. They encode surface proteins responsible for recognizing self versus nonself and play anessential role in the immune response. Consequently, HLA typing is an important test for stem cell and solid organ transplantation, various disease associations, and pharmacogenetics to screen for drug hypersensitivity.HLA mismatches between donors and recipients are the leading cause of graft rejection in tissue and organ transplantation. There is a correlation between the matching based on the precise identification of the HLA allele’s sequences (high-resolution typing) and a decreased risk of rejection. However, the high diversity of the HLA system poses an increased challengefor the unambiguous typing of HLA alleles. Historically, sequence-specific oligonucleotide (SSO) and sequence-specificprimer (SSP) assays have been widely used for HLA typing but fail to achieve high resolution. Alternatively, sequence-based typing (SBT) assays allow the generation of high-resolution results but provide limited throughput and require the utilization of numerous reflexive tests, increasing turnaround time and associated costs.Next-generation sequencing (NGS) technologies triggered a revolution in the HLA typing field by providing the means to generate high-resolution data and resolve most of the allelic ambiguities without the use of multiple reflexive tests.These new technologies allow the simultaneous sequencing of different genomic regions in multiple samples, helping to improve throughput and turnaround time. However, many HLAlaboratories might find NGS challenging to adopt because of the increased complexity, dedicated labor requirements, and high costs. One Lambda™ AllType™ and AllType™ FASTplex™ NGS assays address all of these challenges by offering a robust and simple solution, allowing the parallel high-resolution typing of the HLA-A, HLA-B, HLA-C, DRB1, DRB3, DRB4, DRB5, DQA1,DQB1, DPA1, and DPB1 loci, solving most of the ambiguities and generating data with more than 99% concordance [1]. The AllType FASTplex NGS kit is the only product on the marketfeaturing a single multiplex PCR followed by a one-tube workflow.This feature decreases hands-on and total turnaround time while improving the assay’s reliability by decreasing the number of pipetting steps and the chances of introducing human error. Table 1 summarizes the differences between the AllType and AllType FASTplex NGS kits.AllType FASTplex NGS 11 Loci kit AllType NGS 11 Loci kitTable 1. Specifications for AllType and AllType FASTplex NGS kits.High-performance NGS HLA assays demand high-quality genomic DNA extracted from biological samples such as peripheral blood. With the innovative magnetic beads and reagents included with the Applied Biosystems™ MagMAX™ DNA Multi-Sample Ultra 2.0 Kit, DNA of top quality and purity can be purified for downstream sequencing applications, including HLA typing across various NGS platforms. Combining the MagMAX kit with the Thermo Scientific™ KingFisher™ Apex Benchtop Sample Prep System, the workload is simple and easy while maintaining the flexibility of throughput from 6 to 96 samples. One extraction event can be performed in 45 minutes with only 5 minutes of hands-on time.Here we evaluate the performance of 96 DNA samples extracted from whole blood using the MagMAX kit on the KingFisher Apex system, for high-resolution HLA typing with AllType and AllType FASTplex NGS kits.Experimental procedures Genomic DNA isolationWhole blood was obtained from 96 unique donors in 10 mL acid citrate dextrose (ACD) collection tubes through Tennessee Blood Services. Genomic DNA was extracted from all 96 blood samples using the MagMAX DNA Multi-Sample Ultra 2.0 Kiton the KingFisher Apex system with the 400 µL whole blood workflow. Following extraction, the DNA’s integrity and purity were assessed with a Thermo Scientific™ NanoDrop™ EightSpectrophotometer using A260/A280 and A260/A230 ratios. The integrity of the genomic DNA was analyzed using the GenomicDNA ScreenTape™ Device on the Agilent™ 4200 TapeStation™ System. Concentrations of genomic DNA were obtained using the Invitrogen™ Qubit™ dsDNA BR Assay Kit on the Thermo Scientific™ Varioskan™ LUX Multimode Microplate Reader.HLA typingExtracted genomic DNA from 96 unique blood samples was assayed on Ion Torrent™ and Illumina™ sequencing platforms, for both AllType and AllType FASTplex NGS kits (Table 2, Figure 1).Sample sourceAssay kitSequencing instrumentTable 2. Summary of NGS performed with genomic DNA extracted from 96 unique whole blood samples.12345Collect sampleExtractge NAnomic DAmplify targets and prepare librariesSequenceAnalyze11 Loci kits for Ion Torrent and Illumina platformsFigure 1. Overall workflow for genomic DNA extraction followed by NGS-based HLA typing.AllType and AllType FASTplex NGS assays  thermofisher.com/onelambdaResultsGenomic DNA isolation and qualityNucleic acid extracted from 96 blood samples had concentrations ranging from 57 ng/µL to 365 ng/µL, which are suitable for many downstream sequencing applications (Figure 2). The average A260/A280 ratio across all 96 unique samples was1.89 (Figure 3), indicating highly pure DNA [2]. The integrity ofthe extracted nucleic acid was analyzed using the Genomic DNA ScreenTape Device on the Agilent 4200 TapeStation System. All 96 samples displayed a sharp single band above 51 kb in the genomic DNA region, as shown in the gel image from the Agilent4200 TapeStation System (Figure 4). The DNA integrity number (DIN) was above 9 on average, indicating highly intact genomic DNA [3]. Table 3 shows the genomic DNA quality summary from the 96 unique isolates. Standard deviation (SD) and coefficient of variation (CV) are higher when reporting out concentration yield (ng/µL), because of natural donor variation across the 96samples. Still, smaller SD and CV for DINs and constant A260/A280ratios indicated consistent quality across all 96 samples extracted with the MagMAX kit on the KingFisher Apex system.400Conc. (ng/µL)3503002502001501005000 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96Sample No.Figure 2. Concentrations of genomic DNA from 96 blood samples processed with the MagMAX kit on the KingFisher Apex system. DNA concentrations were obtained using the Qubit dsDNA BR Assay Kit on the Varioskan LUX Multimode Microplate Reader.2.4A260/A280 ratio2.01.61.20.80.400 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96Sample No.Figure 3. Purity of genomic DNA from 96 blood samples processed with the MagMAX kit on the KingFisher Apex system. DNA purity ratios were obtained using the NanoDrop spectrophotometer.Figure 4. Genomic DNA bands from the 96 samples processed with the MagMAX kit on the KingFisher Apex system. DNA bands and profiles were obtained using the Genomic DNA ScreenTape Device on the Agilent 4200 TapeStation System.gDNA concentration (ng/µL)*Size (bp)** DIN score**A260/A280†A260/A230†Table 3. Nucleic acid quality summary for genomic DNA isolated from peripheral blood samples.* Concentration obtained using the Qubit dsDNA BR Assay Kit on the Varioskan LUX Multimode Microplate Reader.** Size and DIN score obtained using the Genomic DNA ScreenTape Device on the Agilent 4200 TapeStation Platform.† Purity and quality absorbance ratios (A260/A280 and A260/A230) were obtained using the NanoDrop spectrophotometer.AllType and AllType FASTplex NGS assays  thermofisher.com/onelambdaQuality and concentrations of amplicons generated with AllType NGS 11 Loci kitsThe quality of the amplicons generated with the AllType NGS 11 Loci kits for Ion Torrent and Illumina sequencing platforms was evaluated using the Agilent 4200 TapeStation System. Expected ranges of approximately 5–6 kb were obtained from all 96 samples (Figure 5). Average concentrations of 55.6 ng/µL and 50.0 ng/µL were obtained forthe Ion Torrent and Illumina platforms, respectively, satisfying the requirements for downstream post-amplification sequencing (Figure 6).LR HLA class I and class II: ~5– 6 kbAllType NGS 11 Loci kit for Ion Torrent platforms AllType NGS 11 Loci kit for Illumina platformsFigure 5. Profile of amplicons generated with AllType NGS 11 Loci kits for Ion Torrent and Illumina platforms and evaluated on the Agilent 4200 TapeStation System. Amplicons were generated from genomic DNA extracted from peripheral blood samples with the MagMAX kit on the KingFisher Apex system. The red triangles indicate approximately 5–6 kb sizes for HLA class I and class II amplicons across all 96 samples.120Kit for Illumina platformsKit for Ion Torrent platforms100Concentration (ng/µL)8060402013579111315171921232527293133353739414345474951535557596163656769717375777981838587899193953 ng/µLSample No.Figure 6. Concentrations of amplicons generated with AllType NGS 11 Loci kits for Ion Torrent and Illumina platforms. Amplicons were generated from genomic DNA extracted from blood with the MagMAX kit on the KingFisher Apex system. Concentrations were measured using the Qubit dsDNA BR Assay Kit. The minimum concentration required for this panel workflow is 3 ng/µL, indicated on the graph by the threshold line; all amplicons exceeded the concentration requirement.AllType and AllType FASTplex NGS assays  thermofisher.com/onelambdaQuality and concentrations of amplicons generated with AllType FASTplex NGS 11 Loci kitsHigh-quality amplicons were also obtained with the AllType FASTplex NGS 11 Loci kits for Ion Torrent and Illumina sequencing platforms. Average length ranges of 5–6 kb were obtained from all samples (Figure 7). Average concentrations of 67.4 ng/µL and 34.2 ng/ µL were obtained for the Ion Torrent and Illumina platforms, respectively, satisfying the requirements for downstream post-amplification sequencing (Figure 8).AllType FASTplex NGS 11 Loci kit for Ion Torrent platforms LR HLA class I and class II: ~5– 6 kb AllType FASTplex NGS 11 Loci kit for Illumina platformsSR exon 1 DRB1: 2.4 kb, DPB1: 2.3 kb, DQB1: 1.5 kbFigure 7. Profile of amplicons generated with AllType FASTplex NGS 11 Loci kits for Ion Torrent and Illumina platforms and evaluated on the Agilent 4200 TapeStation System. Amplicons were generated from genomic DNA extracted from peripheral blood with the MagMAX kit on the KingFisher Apex system. The red triangles indicate approximately 5–6 kb sizes for HLA class I and class II amplicons across all 96 samples. The blue brackets indicate approximately 1.5–2.4 kb sizes for the DRB1, DPB1, and DQB1 amplicons.DNA concentration, AllType FASTplex NGS 11 Loci kits80 Kit for Illumina platformsKit for Ion Torrent platformsConcentration (ng/µL)60402013579111315171921232527293133353739414345474951535557596163656769717375777981838587899193953 ng/µLSample No.Figure 8. Concentrations of amplicons generated with AllType FASTplex NGS 11 Loci kits for Ion Torrent and Illumina platforms. Amplicons were generated from genomic DNA extracted from peripheral blood with the MagMAX kit on the KingFisher Apex system. Concentrations were measured using the Qubit dsDNA BR Assay Kit. The minimum concentration required for this panel workflow is 3 ng/µL, indicated on the graph by the threshold line; all amplicons exceeded the concentration requirement.AllType and AllType FASTplex NGS assays  thermofisher.com/onelambdaSequencing performance of amplicons generated with AllType and AllType FASTplex NGS kitsPost-amplification sequencing was performed on both Ion Torrent and Illumina platforms. The average read depth (Table 4) was estimated using One Lambda™ TypeStream™ Visual NGS Analysis Software 3.0 and used as quality metrics across all regions of the 11 HLA loci. The minimum recommended read depth for both AllType and AllType FASTplex NGS kits is 100. This requirement was surpassed with both kits and sequencing platforms (Table 4).HLA genotypes were automatically estimated by TypeStream Visual NGS Analysis Software, and the accuracy of the test was determined by comparing results obtained with both AllType and AllType FASTplex NGS kits on the Ion Torrent and Illumina platforms. No discrepancies (i.e., concordance of 100%) were observed across both kits and both sequencing platforms, as expected when high-quality genomic DNA is used as initial input for both the AllType and AllType FASTplex NGS kits.ConclusionAutomated DNA extraction using the MagMAX DNA Multi- Sample Ultra 2.0 Kit on the KingFisher Apex system consistently generated high-quality genomic DNA, suitable for downstream HLA typing with AllType and AllType FASTplex NGS kits on both Ion Torrent and Illumina NGS platforms. Sequencing analysis with individual allele assignments can be automatically generated by TypeStream Visual NGS Analysis Software and can provide results in less than 3 minutes.Table 4. Average read depth for all regions across 11 HLA loci after sequencing performed with AllType and AllType FASTplex NGS kits on Ion Torrent and Illumina platforms.AllType and AllType FASTplex NGS assays  thermofisher.com/onelambdaSample isolation reagents and equipmentCat. No.ProductOrdering informationMagMAX DNA Multi-Sample Ultra 2.0 Kit A36570KingFisher Apex Benchtop Sample Prep System with 96 Deep-Well Head 5400930HLA typing reagentsAllType NGS 11 Loci Sample Prep Flex Kit for Ion Torrent platforms ALL-PREP11LXAllType NGS 11 Loci Sample Prep Flex Kit for Illumina platforms ALL-PREP11LFXAllType FASTplex NGS 11 Loci Kit for Ion Torrent platforms ALL-FAST11LXAllType FASTplex NGS 11 Loci Flex Kit for Illumina platforms ALL-FAST11LFXQuality control reagents and equipmentNanoDrop Eight Spectrophotometer 840-343700Varioskan LUX Multimode Microplate Reader VL0000D0Qubit dsDNA BR Assay Kit Q32850ReferencesAuthorsHarry Lopez, Lillie Manley, Michelle Leija, Peter Brescia, Rodrigo Francisco, Jim George, Thermo Fisher Scientific. Learn more at For Research Use Only. Not for use in diagnostic procedures. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Agilent, TapeStation, and ScreenTape are trademarks of Agilent Technologies, Inc. Illumina, MiSeq, MiniSeq, and iSeq are trademarks of Illumina Inc. COL35639 0523