Next-generation Sequencing Library Preparation Research Articles

A Method for Detection of Somatic LINE-1 Insertions at the Single-Cell Level from Postmortem Human Brain.

Long Interspersed Element-1 (LINE-1, L1) is a retrotransposon that has the ability to amplify its copy in the genome autonomously. L1Hs is a human-specific active subtype of L1 reported to amplify its copy in neural progenitor cells causing genomic mosaicism. This chapter describes a new method named NECO-seq (Novel Elements Concentrated-sequence) to identify the genomic locus of L1Hs insertions at the single-cell level. This protocol contains the steps of (1) preparation of neuronal cell nuclei from a postmortem human brain, (2) whole genome amplification from single neural nuclei (snWGA), (3) single nucleotide polymorphisms (SNPs) genotyping for quality control of snWGA products, (4) library preparation for next-generation sequencing to enrich the genomic locus of L1Hs insertions, and (5) bioinformatic analysis to detect novel somatic L1Hs insertions. This method can detect approximately 97% of L1Hs originally existing in reference human genome and approximately 10-20 newly inserted L1Hs copies in a neuronal cell of a postmortem human brain.

Illuminating the Artefacts of NGS Seen in PGT-A & PGT-SR

Background: Advancements in next generation sequencing (NGS) based genomics over the past 10 years have allowed the routine adoption of preimplantation genetic testing for aneuploidy (PGT-A), and/or segmental rearrangements (PGT-SR) of [Formula: see text]10Mb 1 in the assisted reproductive technology (ART) clinic. However, one challenge remains within the assessment of aligned short read sequencing data, in the ability to discriminate between embryonic profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification or library preparation. Aim: To identify the common artefacts seen in next generation sequencing (NGS) based PGT to reduce inconclusive or false positive results. Method: NGS profiles generated from biopsied human embryonic trophectoderm cells were manually analysed for common genomic artefacts. Following trophectoderm biopsy within a clinical ART cycle, cell samples underwent DNA amplification (Illumina SureplexTM DNA amplification system), sequencing (VeriSeqTM PGS kit on the MiSeq[Formula: see text] System, and bioinformatic data analysis (Bluefuse Multi v4.4, Illumina). Results: Common artefacts were identified affecting chromosomes 7, 11, and 19. Artefacts imitated small CNVs typically displaying a [Formula: see text]50% increase in centromeric sequence counts on chromosomes 7 and 11, and a global increase of sequence counts of [Formula: see text]40% across the entire chromosome 19 (except at the centromere, presenting as diploid). Interestingly a technical repeat of the library preparation and sequencing of the original DNA template somewhat normalised these artefacts. Conclusion: The study found three commonly occurring artefacts involving chromosomes 7, 11, and 19 artificially introduced during suboptimal NGS library preparation. Importantly these artefacts may be normalised through technical repeat of library preparation and sequencing. The awareness of these artefacts may reduce false positive and/or inconclusive results, increasing clinical embryonic utilisation following PGT.

Quality Control of Biospecimens in a Danish Clinical Cytology Biobank.

Introduction: Based on the experience from a Swedish biobank, we established a clinical cervical cytology biobank and adapted it to a Danish setting. The aim of the present study was to validate the biobank material regarding quality and quantity, to determine the usefulness of the material for future diagnostics and biomarker testing. Methods: Cervical cytology samples collected in ThinPrep were analyzed before and after biobanking using p16/ki-67 dual staining, a human papillomavirus (HPV) DNA test (Cobas), and a test for HPV messenger RNA (mRNA; Aptima). The concordance of the test results before and after biobanking was assessed. We also evaluated the morphology before and after biobanking and did additional tests on the biobanked material to qualify the usefulness of the material (library preparation for next-generation sequencing [NGS], reverse transcription-polymerase chain reaction [RT-PCR], and the Inno-Lipa HPV genotyping test). Results: For the Cobas HPV test, the concordance was 92% (122/133), and for the Inno-Lipa test (30 samples), it was 100%. For the Aptima assay, the concordance was a little lower, 84% (42/50). The morphology of the cell was well preserved, and the concordance of the p16/ki-67 dual staining was 88% (37/42). The functional tests showed that DNA-based NGS libraries (TST15 panel; Illumina) had good quality parameters. However, with the RT-PCR, 12% of the samples showed poor quality and a too low input amount for the analysis. Conclusion: The quality of the biobanked samples is high, and the material is suitable for testing of DNA, RNA, and protein. However, for testing of specific biomarkers, pilot studies are recommended to ensure sufficient input amount and quality of the material, especially for RNA-based studies.

Non-target RNA depletion strategy to improve sensitivity of next-generation sequencing for the detection of RNA viruses in poultry.

PCR-based assays have become the benchmark for detecting pathogens of poultry and other livestock; however, these techniques are limited in their ability to detect multiple infecting agents, provide limited genetic information on the pathogen, and, for RNA viruses, must be reviewed frequently to assure high sensitivity and specificity. In contrast, untargeted, high-throughput sequencing can rapidly detect all infecting agents in a sample while providing genomic sequence information to allow more in-depth characterization of viruses. Although next-generation sequencing (NGS) offers many advantages, one of its primary limitations is low sensitivity to pathogens given the abundance of host and other non-target sequences in sequencing libraries. We explored methods for improving the sensitivity of NGS to detect respiratory and enteric viruses in poultry from RNA extracts of swab samples. We employed commercial and custom-designed negative enrichment strategies to selectively deplete the most abundant rRNA reads from the host and non-target bacteria; host RNA was diminished from up to 40% of total reads to as low as 3%, and the total number of reads assigned to abundant bacterial classes were reduced greatly. Our treatment resulted in up to a 700-fold increase in the number of viral reads, detection of a greater number of viral agents, and higher average genome coverage for pathogens. Depletion assays added only 2 h to the NGS library preparation workflow. Custom depletion probe design offered significant cost savings (US$7-12 per sample) compared to commercial kits (US$30-50 per sample).

Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Fragmentation assessment of FFPE DNA helps in evaluating NGS library complexity and interpretation of NGS results

Formalin-fixed paraffin-embedded (FFPE) tissue remains the most common source for DNA extraction from human tissue both in research and routine clinical practice. FFPE DNA can be considerably fragmented, and the amount of DNA measured in nanograms may not represent the amount of amplifiable DNA available for next-generation sequencing (NGS). Two samples with similar input DNA amounts in nanograms can yield NGS analyses of considerably different quality. Nevertheless, many protocols for NGS library preparation from FFPE DNA describe input DNA in nanograms without indication of a minimum requirement of amplifiable genome equivalent DNA.An important NGS quality metric is the library complexity, reflecting the number of DNA fragments from the original specimen represented in the final library. Aiming to illustrate the relationship between DNA fragmentation degree and library complexity, we assessed the fragmentation degree of 116 lung cancer FFPE DNA samples to calculate the amount of amplifiable input DNA used for library preparation. Mean unique coverage, coverage uniformity, and mean number of PCR duplicates with the same unique molecular identifier were used to evaluate library complexity.We showed that the amount of amplifiable input DNA predicted library complexity better than the input measured in nanograms. The frequent discrepancy between DNA amount in nanograms and the amount of amplifiable DNA indicate that the fragmentation degree should be considered when performing NGS of FFPE DNA. Importantly, the fragmentation assessment may help when interpreting NGS data and be a useful tool for evaluating library complexity in the absence of unique molecular identifiers.

Advanced preparation of fragment libraries enabled by oligonucleotide-modified 2\u2032,3\u2032-dideoxynucleotides

The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2′,3′-dideoxynucleotide (ddONNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddONNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddONNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.

EP376: NGS detection of NUDT15 6-bp insertion and UGT1A1 (TA) repeat polymorphism on a preventative genomics assay

Application of Next-Generation Sequencing (NGS) for pharmacogenetic analysis provides cost-effective and high-throughput alternatives to other genotyping methodologies, making it a method of choice for pharmacogenetic testing. However, the nature of short-read sequencing technologies may carry challenges when it comes to the detection of specific complex structures/variants such as large insertion/deletions (indels), or repeat polymorphisms. These limitations can be overcome by the design of sequencing probes and optimized NGS library preparation process, as well as customized bioinformatic analysis pipelines. UGT1A1 is an important pharmacogene that impacts response to multiple drugs, including the HIV treatment, Atazanavir. The TA repeat polymorphism, rs3064744, is identified across various geographic, ethnic and racial populations and causes reduction in gene activity, resulting in toxicity and dosage effects. NUDT15 plays an important role in the metabolism of thiopurines. The rs746071566 microsatellite polymorphism, c.38GAGTCG[4] (p.13GV[4]), plays a critical role in thiopurine-induced adverse reactions and has been identified as a commonly observed variant in pan-ethnic populations. The current study evaluated the performance of a targeted NGS capture and bioinformatics platform to identify the promoter (TA)7 repeat polymorphism UDP glucuronosyl-transferase UGT1A1, as well as a 6 bp insertion NUDT15 variant. Both variants were analyzed as a part of a 455 gene panel utilized for a preventative genomics assay. The study consisted of 413 validation samples analyzed for UGT1A1 (rs3064744), and 186 analyzed for NUDT15 (rs746071566). The specimens were retrieved from various sources: DNA from Coriell Repository, and DNA isolated from whole blood and saliva by the Maxwell RSC Blood DNA Kit (Promega, Madison, Wisconsin) on the automated Maxwell RSC 48 Instrument (Promega). Sequencing was performed using custom-designed targeted capture and Fast Hybridization Target Enrichment workflow (Twist Bioscience, South San Francisco, CA). The samples were prepared using 50 ng of input DNA and the Twist EF Library Prep Kit 2.0 + UDIs and Fast Hyb reagents (Twist Bioscience), and run in a format of 12 samples per pool and 36 per single run on MiniSeq System (Illumina, San Diego, CA). Selected samples were confirmed using Sanger sequencing. The data was analyzed using the Edico Dragen server (Illumina), and in-house developed proprietary pipeline. For the purpose of this study, the reference ranges were defined for UGT1A1 as *1 AT[7], *28 AT[8], *36 AT[6] and *39 AT[9]. The genotypes frequency detected for UGT1A1 rs3064744 were as follow: AT[7]/AT[6] 0.97%, AT[7]/AT[7] 62%, AT[7]/AT[8] 24%, AT[7]/AT[9] 0.97%, AT[8]/AT[8] 11.8%, AT[9]/AT[9] 0.24%; and for NUDT15 rs746071566 GGAGTC[1]/GGAGTC[1] 99.5%, GGAGTC[1]/GGAGTC[2] 0.5% and GGAGTC[1]/GGAGTC[0] 0%. This data is comparable to the frequency published in the gnomAD database. Additionally, Sanger confirmation analysis performed for selected Coriell samples yielded 100% concordance between NGS and Sanger sequencing data. As previously reported, ∼14% of variants (eg, indels, small CNVs, and complex changes) identified during NGS analysis can impose technical difficulties resulting in decreased detection rates. Both of the variants investigated in this study (UGT1A1 rs3064744 and NUDT15 rs746071566) belong to this group of challenging variants, and thus require specific consideration during the analytical and bioinformatic design and validation. The NGS capture presented in this study was iteratively designed to optimize the ability to sequence across these regions. Performance data for these variants is dependent on wet laboratory procedure and bioinformatic variant assembly because standard VCF calling software packages do not account for repetitive elements. The in-house developed bioinformatic script accurately translated the output of the VCF pipeline into the genotype calls. The comparability of the obtained genotype frequencies with the published data in gnomAD further confirmed the accuracy of the assay performance. Analysis of the samples using Sanger sequencing further confirmed data received on NGS analysis.

Abstract P2-01-15: Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance

Abstract Introduction: Growing data established the importance of monitoring dynamic changes in circulating tumor DNA (ctDNA) to identify early signs of therapeutic responses, allowing for timely management of treatment to achieve more effective personalized therapy. Higher assay accuracy and consistency, and lower assay cost will support more clinical validation trials and benefit more cancer patients with non-invasive ctDNA NGS tests that can simultaneously map multiple genomic alterations at an affordable price. Method: The NVIGEN X - Precision Cancer Profiling test is a next generation sequencing (NGS) based circulating tumor DNA detection assay using the hybridization capture approach with customized gene panels. Our ctDNA NGS assay was developed with the use of high performance magnetic nanobeads, which enhances assay workflow at key steps including cfDNA extraction, NGS library preparation, and target enrichment. Experiments with individual plasma samples and DNA mutant fragments spiked in plasma samples were carried out to establish the assay performance such as sensitivity, specificity, consistency and data efficiency. NGS data QC metrics of the NVIGEN assay were compared with other assays in peer reviewed publications. Results: We developed a focus 32 gene panel that covers 144 kb of gene regions of clinical significance for breast cancer treatment monitoring and guidance, such as AKT1, ERBB2, PIK3CA, EGFR, ESR1, BRCA1/2, and CD274. Our results demonstrated the capability of NVIGEN X ctDNA NGS assay to detect rare copies (8 cp) of gene mutation at 0.07% MAF from DNA mutant fragments spiked into plasma samples. The NVIGEN X ctDNA NGS assays consistently presented 2-5% duplication rate, >80% on-target rate, <10% CV for key NGS data metrics, and on average required 1.36X paired reads per 1X unique coverage. Compared with the Roche Avenio assays (targeted, expanded and surveillance panels) as published in 2020 which on average required 9.36X paired reads per 1X unique coverage, the NVIGEN X -precision cancer profiling assays demonstrated 85% reduction in NGS data need to generate each unique coverage. Compared with the original Capp-seq data as published in the 2014 Nature Medicine paper which required in average 13.78 or 27.56 paired reads per unique coverage, the NVIGEN X assay demonstrated >90% reduction in NGS data need per unique coverage. Conclusion: The NVIGEN X - Precision Cancer Profiling assay provided high NGS assay performance with high sensitivity, specificity, and consistency, and significantly improved NGS data efficiency. This allows for dramatically reduced assay cost and will help support routine applications of ctDNA NGS tests to improve cancer patient treatment. Experiments of applying NVIGEN X assays for clinical research with patient samples are ongoing and will be presented. Citation Format: Aihua Fu, Wenwu Cui, Minh V. Ton, Kevan Wang, Weiwei Gu, Tianhong Li, Heather A. Parsons, Minetta C. Liu, George W. Sledge. Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-15.

Optimization of enzymatic fragmentation is crucial to maximize genome coverage: a comparison of library preparation methods for Illumina sequencing

BackgroundNovel commercial kits for whole genome library preparation for next-generation sequencing on Illumina platforms promise shorter workflows, lower inputs and cost savings. Time savings are achieved by employing enzymatic DNA fragmentation and by combining end-repair and tailing reactions. Fewer cleanup steps also allow greater DNA input flexibility (1 ng-1 μg), PCR-free options from 100 ng DNA, and lower price as compared to the well-established sonication and tagmentation-based DNA library preparation kits.ResultsWe compared the performance of four enzymatic fragmentation-based DNA library preparation kits (from New England Biolabs, Roche, Swift Biosciences and Quantabio) to a tagmentation-based kit (Illumina) using low input DNA amounts (10 ng) and PCR-free reactions with 100 ng DNA. With four technical replicates of each input amount and kit, we compared the kits’ fragmentation sequence-bias as well as performance parameters such as sequence coverage and the clinically relevant detection of single nucleotide and indel variants. While all kits produced high quality sequence data and demonstrated similar performance, several enzymatic fragmentation methods produced library insert sizes which deviated from those intended. Libraries with longer insert lengths performed better in terms of coverage, SNV and indel detection. Lower performance of shorter-insert libraries could be explained by loss of sequence coverage to overlapping paired-end reads, exacerbated by the preferential sequencing of shorter fragments on Illumina sequencers. We also observed that libraries prepared with minimal or no PCR performed best with regard to indel detection.ConclusionsThe enzymatic fragmentation-based DNA library preparation kits from NEB, Roche, Swift and Quantabio are good alternatives to the tagmentation based Nextera DNA flex kit from Illumina, offering reproducible results using flexible DNA inputs, quick workflows and lower prices. Libraries with insert DNA fragments longer than the cumulative sum of both read lengths avoid read overlap, thus produce more informative data that leads to strongly improved genome coverage and consequently also increased sensitivity and precision of SNP and indel detection. In order to best utilize such enzymatic fragmentation reagents, researchers should be prepared to invest time to optimize fragmentation conditions for their particular samples.

Monitoring Chromatin Regulation in Planarians Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq).

Planarians are an accessible model system to study animal regeneration and stem cells. Over the last two decades, new molecular techniques have provided us with powerful tools to understand whole-body regeneration and pluripotent adult stem cells specifically. We describe a method for performing Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) on planarian cells that relies on FACS to isolate different cell populations followed by immunoprecipitation and library preparation for next-generation sequencing. Whole-genome profiling of histone modifications enables a greater understanding of epigenetic mechanisms in development, pluripotency, and differentiation. This protocol adds to the growing list of functional genomic approaches to study whole-body regeneration in animals.

POLYseq: A poly(β-amino ester)-based vector for multifunctional cellular barcoding.

SummaryDespite evolving biological application of next-generation sequencing (NGS) at single-cell level, current techniques in NGS library preparation restrict multiplexing, necessitating the costly preparation of distinct libraries for each sample. Here, we report the development of a novel poly(β-amino) ester labeling system synthesized with inexpensive, common reagents, termed POLYseq, capable of efficiently delivering fluorescent molecules or sample-distinguishing DNA barcodes through non-covalent binding enabling rapid creation of custom sample pools. Chemical formulation was found to determine cellular labeling propensity. Live image-based tracking of fluorescent conjugated POLYseq vectors demonstrated lysosomal compartmentalization. Barcode labeling was uniformly detected across 90% of cells by single-cell RNA sequencing, allowing for the successful identification of human and mouse cultured cell lines from a single pool. These findings highlight the multifunctional applications of POLYseq in live cell imaging and NGS in a scalable and cost-effective manner.

Abstract 707: Detection of SARS-CoV-2 co-infections with seasonal respiratory viruses using a multiplexed amplicon NGS panel

Abstract The ongoing COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected everyone around the world. Most symptoms of COVID-19 overlap with symptoms of the flu and the common cold. With the approaching flu season, early and accurate detection of these viruses are critical not only to slow the spread of SARS-CoV-2, but also to ensure that patients receive the appropriate care based on their diagnosis, and to keep the healthcare system from becoming overloaded. We designed a next-generation sequencing (NGS) assay to detect the SARS-CoV-2, influenza A H1N1, influenza A H3N2, influenza B, RSV A, and RSV B viruses, and to allow for surveillance and subtype identification. The primers for this multiplex PCR-based method interrogate nearly every gene of each virus. Subsequent sequencing enables not only virus detection but also co-infection detection for these respiratory viruses. In addition, sequencing of virus-positive samples makes it possible to use this assay for mutation and strain analysis. Using the CleanPlex target enrichment technology, we prepared targeted amplicon libraries with human total RNA (50ng) spiked with SARS-CoV-2 synthetic RNA, influenza A synthetic RNA, influenza B synthetic RNA, RSV A RNA, and RSV B RNA (100-10,000 viral genome copies), either individually or in combination. The NGS library preparation protocol includes a reverse transcription step to generate cDNA, followed by a multiplexed PCR step for specific amplification of viral targets. A critical subsequent background cleaning step removes non-specific PCR products, and a final PCR adds sample indices and Illumina sequencing adapters to complete the NGS library preparation. Libraries were sequenced on the Illumina iSeq100 platform, and sequencing metrics as well as virus characterization were determined using Paragon Genomics's bioinformatics pipeline. Each sample was sequenced at 1,000 average paired-end reads per amplicon. After aligning sequencing reads to each viral genome, we were able to detect nearly every gene of each virus with high specificity. At viral loads of 10,000 copies, 96-100% of amplicons were covered at >100X, and mapped to the intended virus species as well as to the intended subtypes for influenza A and RSV. The assay also accurately identified co-infected samples, which contained equal copies of SARS-CoV-2 and influenza (either A H1N1, A H3N2, or B). In summary, the assay detects multi-target respiratory viruses with high accuracy and specificity. We plan to expand on our findings for this assay's performance with additional targets, co-infection studies and mutational analysis studies. Citation Format: Lucie S. Lee, Rounak Feigelman, David Debruyne, Julia Spencer, Chenyu Li, Zhitong Liu, Guoying Liu. Detection of SARS-CoV-2 co-infections with seasonal respiratory viruses using a multiplexed amplicon NGS panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 707.

Abstract 2286: Use of a synthetic spike-in ladder to measure NGS library complexity

Abstract Background: Detection of rare variants in traditional and liquid biopsies is becoming increasingly important for cancer diagnosis, monitoring and treatment. Next Generation Sequencing (NGS) has the capacity to detect known and novel variants in small biological samples with a high level of sensitivity. However, the limit of detection is affected by sample DNA concentration and integrity, technical error and number of unique molecules captured and sequenced (library complexity). Unique molecular identifiers (UMl) and random fragment end analysis commonly are used to measure complexity yet both are reported to have systematic biases leading to a non-random read distribution when measured, altering interpretation of results and potentially skewing data. Here we describe the use of a spike in complexity calibration ladder comprising synthetic DNA internal standard competitors (IS) as an orthogonal measure of library complexity. Methods: An Endogenous Complexity Calibration Ladder (ECCL) comprising multiple unique synthetic IS at different concentrations was created. All ECCL IS share homology with each other and endogenous human SCGB1A1 sequence but each contain nucleotide changes at different positions along the sequence string so that they can be distinguished. The ECCL was mixed with several IS targeting different TP53 exons in a fixed proportion. The ECCL/TP53 IS mixture is designed to be used in either amplicon or hybrid-capture library preparation. Here, amplicon libraries were generated. Molecule numbers and ratios between IS were determined using deep sequencing of multiple replicates. The ECCL/TP53 IS mix was spiked into a commercial human gDNA prior to NGS library preparation. Serial dilutions (1.5-, 3-, 6-, 12- and 24-fold) and intentional addition of inhibitors were conducted to stress and test the system. The ECCL/TP53 IS mixture also was spiked into gDNA from 60 primary airway epithelial cell (AEC) specimens prior to library preparation and NGS to measure TP53 variant fraction while controlling for complexity. Results: Observed complexity measured using the ECCL was close to expected for each serially diluted mixture of gDNA and ECCL/TP53 IS. The precision of complexity measurement for less dilute and less inhibited samples was limited by absence of finer titration of the least concentrated IS in ECCL. Use of ECCL enabled measurement of inter-sample variation in complexity among the 60 AEC specimens. Conclusions: The ECCL controls for both sample and library specific variation in complexity, and enables better estimation of the lower limit of detection for variant allele fraction (VAF). Additionally, it is compatible with the use of target specific IS which allow for improved measurement of technical sequencing error. The variation in complexity observed in primary AEC gDNA samples demonstrates the need to account for this when assessing rare variant allele fraction. Citation Format: Erin L. Crawford, Tian Chen, Daniel J. Craig, James C. Willey. Use of a synthetic spike-in ladder to measure NGS library complexity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2286.

Challenges in the application of NGS in the clinical laboratory

Next-generation sequencing (NGS), also known as massively parallel sequencing, has revolutionized genomic research. The current advances in NGS technology make it possible to provide high resolution, high throughput HLA typing in clinical laboratories. The focus of this review is on the recent development and implementation of NGS in clinical laboratories. Here, we examine the critical role of NGS technologies in clinical immunology for HLA genotyping. Two major NGS platforms (Illumina and Ion Torrent) are characterized including NGS library preparation, data analysis, and validation. Challenges of NGS implementation in the clinical laboratory are also discussed, including sequencing error rate, bioinformatics, result interpretation, analytic sensitivity, as well as large data storage. This review aims to promote the broader applications of NGS technology in clinical laboratories and advocate for the novel applications of NGS to drive future research.

Isolation of target DNA using synergistic magnetic bead transport and electrokinetic flow.

The advent and dissemination of next-generation sequencing (NGS) technologies such as Illumina's sequencing platforms has brought forth vast reductions in the cost, time, and technical difficulties associated with DNA and RNA sequencing. Despite this trend, the workflow required to generate nucleic acid libraries for sequencing remains time-consuming and laborious. The following research proposes a method for simplifying and streamlining this process by replacing the manual washing steps of the common magnetic bead-based cleanup with a novel microfluidic method by integrating magnetic separation and electrokinetic purification (MSEP). Requiring no pumps, pipette mixing, vortexing, or centrifugation, MSEP relies on selective adsorption of target DNA onto the magnetic beads with subsequent transport of beads through a microchannel undergoing an antiparallel electroosmotic flow. The synergetic flow conditions were optimized using a simple electrohydrodynamic flow model. This work demonstrates that MSEP is as effective in eliminating adapter-dimers from the post-ligation library mix as the manual method while also greatly reducing the hands-on time and amount of pipetting required. Although MSEP has been applied specifically toward NGS library preparation at this time, it has the potential to be adapted and employed for any bead-based separation scheme, namely, solid phase extraction, sequence-specific hybridization, and immunoprecipitation on a microscale.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq) is a new ultra-sensitive UMI-based NGS library preparation technology for use with cfDNA and cfRNA

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20–25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

An archaeogenetic approach to identify the remains of the Hungarian Kings. Working Plan

The Royal Basilica of Székesfehérvár was the burial place of fifteen Hungarian kings. Unfortunately, the anthropological findings excavated at the site of the Basilica were mixed up during the tumultuous centuries of Hungary, hence the royal remains still lie unidentified in a charnel-house. The appearance and rapid development of archaeogenetics now allows the personal identification of the royal skeletons from among the remains of the Basilica. The genetic information necessary for the identification of the Árpád dynasty members is accessible, while sequence data of the non-Árpádian kings’ relatives still need to be obtained by further genetic analysis. Here we provide an outline of the investigation for the identity of the royal skeletons: we sketch the process of sample preparation and DNA extraction, the steps of library preparation for next-generation sequencing (NGS) and give a brief report of the current progressions.

423. SARS-CoV-2 NGS Assay Powered by Biotia COVID-DX Software

BackgroundCOVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome.MethodsThe SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis.ResultsThe SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens.ConclusionThe SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease.DisclosuresDorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

Identification of differentially methylated HpaII sites by NGS analysis of HpaII-digested libraries

Differentially methylated regions (DMRs) have been widely explored as epigenetic biomarkers. Here, we developed a novel approach combining methylation-sensitive restriction enzyme (MSRE) and next-generation sequencing (NGS) to identify DMRs between chorionic villi (CV) and maternal blood cells (MBC). During NGS library preparation, adapter-ligated genomic DNA of CV and MBC were digested with the MSRE, HpaII, and PCR-amplified. As unmethylated HpaII sites were cleaved, the resulted library should contain only methylated HpaII sites. By sequencing both HpaII-digested CV and MBC libraries, 9 differentially methylated-HpaII sites on chromosome 21 which exhibited more than 50% methylation increase in CV were identified. These DMRs are epigenetic biomarkers to tell the difference between CV and MBC. Our approach will also be applicable to screen various tissue-specific epigenetic biomarkers.