Next-generation Sequencing Library Preparation Research Articles

Leveraging the fundamentals of heat transfer and fluid mechanics in microscale geometries for automated next-generation sequencing library preparation

Next-generation sequencing (NGS) is emerging as a powerful tool for molecular diagnostics but remains limited by cumbersome and inefficient sample preparation. We present an innovative automated NGS library preparation system with a simplified mechanical design that exploits both macro- and microfluidic properties for optimizing heat transfer, reaction kinetics, mass transfer, fluid mechanics, adsorption–desorption rates, and molecular thermodynamics. Our approach introduces a unique two-cannula cylindrical capillary system connected to a programmable syringe pump and a Peltier heating element able to execute all steps with high efficiency. Automatic reagent movement, mixing, and magnetic bead-based washing with capillary-based thermal cycling (capillary-PCR) are completely integrated into a single platform. The manual 3-h library preparation process is reduced to less than 15 min of hands-on time via optimally pre-plated reagent plates, followed by less than 6 h of instrument run time during which no user interaction is required. We applied this method to two library preparation assays with different DNA fragmentation requirements (mechanical vs. enzymatic fragmentation), sufficiently limiting consumable use to one cartridge and one 384 well-plate per run. Our platform successfully prepared eight libraries in parallel, generating sequencing data for both human and Escherichia coli DNA libraries with negligible coverage bias compared to positive controls. All sequencing data from our libraries attained Phred (Q) scores > 30, mapping to reference genomes at 99% confidence. The method achieved final library concentrations and size distributions comparable with the conventional manual approach, demonstrating compatibility with downstream sequencing and subsequent data analysis. Our engineering design offers repeatability and consistency in the quality of sequence-able libraries, asserting the importance of mechanical design considerations that employ and optimize fundamental fluid mechanics and heat transfer properties. Furthermore in this work, we provide unique insights into the mechanisms of sample loss within NGS library preparation assays compared with automated adaptations and pinpoint areas in which the principles of thermodynamics, fluid mechanics, and heat transfer can improve future mechanical design iterations.

Abstract 317: Using the Biomek NGeniuS Next Generation Library Prep System to automate the Illumina TruSight Oncology 500 assay

Abstract Cancer researchers using next generation sequencing (NGS) need reliable sample preparation methodologies that generate high-quality, sequence-ready libraries from often-challenging samples. This process can be aided using automated liquid handling systems. The Biomek NGeniuS Next Generation Library Prep System from Beckman Coulter Life Sciences is a reliable, easy-to-use, purpose-built liquid handler for NGS library preparation ideal for laboratories requiring more flexibility and less hands-on time. The user-friendly software requires no programming skills, making it easy to set up and run. Biomek NGeniuS Next Generation Library Prep System’s hardware features include an on-deck thermal cycler, gripper, temperature-controlled reagent storage, and high-density tip storage which reduces user interventions and enables more walkaway time. An extensive and growing library of complimentary demonstrated NGS sample prep protocols allows the user to select from a variety of applications for NGS workflows. One such workflow is Illumina TruSight Oncology 500 assay, which represents a comprehensive NGS assay targeting the full coding regions of 523 genes implicated in the pathogenesis of solid tumors. Using enrichment-based library preparation techniques for use with formalin-fixed, paraffin-embedded (FFPE) samples, the Illumina TruSight Oncology 500 assay, when running the DNA Only workflow, can detect single nucleotide variants (SNVs), indels, amplifications, and multinucleotide variants (MNVs) in a single sequencing run. The Illumina TruSight Oncology 500 assay also detects immunotherapy biomarkers for tumor mutational burden (TMB) and microsatellite instability (MSI) in DNA samples. The Illumina TruSight Oncology 500 assay also offers a combined RNA/DNA workflow that also allows for the assessment of fusion events. In this poster, we show the utility of the Biomek NGeniuS Next Generation Library Prep System to perform the Illumina TruSight Oncology 500 assay, which saves researchers valuable hands-on time and offers a highly streamlined setup process for users new to liquid handling automation. Confidential - Company Proprietary Citation Format: Zach Smith. Using the Biomek NGeniuS Next Generation Library Prep System to automate the Illumina TruSight Oncology 500 assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 317.

Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome

BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation “blacklist” in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

Elevating the Educational Mission of "Full-Service" Core Facilities through Formal Biotechnology Workshops.

Core facility laboratories are an essential part of the successful research enterprise of many universities around the world. Core facilities provide state-of-the-art instrumentation and technologies to support research of all faculty, postdocs, and students on a fee-for-service basis. Academic next-generation sequencing cores are typically "full service" facilities, and access to and training on their instrumentation is limited to core staff. To address these limitations, we provided graduate students with technical training at our core facility. We developed a 1-week noncredit-bearing workshop and recruited 6 graduate students (N = 6) as part of a pilot program. The program involved online teaching, classroom-based teaching, and hands-on training in next-generation sequencing library preparation and sequencer operation. A post-participation survey revealed highly positive outcomes in terms of skill development and increased awareness of technologies offered by the core facility. A workshop of this scale could be incorporated into the graduate curriculum and extended to core facilities that focus on other technologies. We believe that introducing formal standardized teaching spearheaded by core facilities would improve the graduate student curriculum and hope that this study can provide guidance on curriculum design for similar workshops.

#249 : Spotlight on the Artefacts in Next Generation Sequencing in PGT-A: Possible Reason for High Mosaicism Reporting

Background and Aims: Advancement in Next Generation Sequencing (NGS) has led to the extensive utilization of Preimplantation Genetic testing for Aneuploidy. But one challenge, in reporting of aligned short read sequencing data, is the ability to discriminate between embryonic NGS profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification/library preparation. Do technical errors in VeriSeq-NGS lead to artefacts in CNV charts resulting in falsely increased reporting of chromosomal mosaicism? Method: After ethical clearance and informed consent, patients [Formula: see text] 35 years, [Formula: see text]1 implantation failures and severe male factor infertility underwent PGT-A. Those with aneuploid embryos on TE biopsy, donated their embryos for research in year 2022. The DNA extraction and amplification was carried out using Sureplex-DNA amplification system. The libraries were prepared using VeriSeqTM-PGS Library Preparation kit. A total of 24 libraries were pooled, denatured and subjected to NGS using Illumina MiSeq system. CNV visualization and analysis for each sample was carried out using BlueFuse Multi Software (Illumina, USA). The aneuploid whole blastocyst were subjected to the same protocol. Results: NGS profiles generated from the aneuploid whole blastocysts, were manually analyzed for CNVs and for any common genomic artifacts. The quality control was performed using Illumina-Quality Control requirements and only samples satisfying these requirements were used for analysis in the study. Common artifacts were identified affecting chromosome 7,11,16 and 19. Out of 22 blastocysts, NGS profiles of 21 blastocysts had artifact at chromosome 19, and 15 had artifacts at chromosome 7,11,16. These artifacts may be introduced during whole genome amplification and/or suboptimal NGS library preparation or is inherent weakness of the VeriSeq-NGS kit. Conclusion: The four commonly occurring artefacts involved chromosome 7,11,16 and 19, which could be introduced during steps of whole genome amplification or library preparation. The awareness of these artefacts is important to reduce the false positive/inconclusive rate.

B-016 Automated Next Generation Sequencing Fluidic-Based Device for Sample to Library Preparation: A Flexible Solution for Rapid Decentralized Sequencing for Infectious Disease Diagnostics

Abstract Background In addition to the established use of Next Generation Sequencing (NGS), such as drug resistance monitoring in HIV, the recent COVID19 pandemic has shown the crucial need of NGS technologies as tools for identification of clinically relevant variants of the SARS-CoV-2 virus. Typically, in these settings, to be cost-effective most NGS analyses were conducted using larger batch sizes often resulting in long turn-around times of 2–3 weeks the need of trained personnel, and centralized testing facilities. Methods We have developed an easy to use automated fluidic-based benchtop device to bridge a perceived gap in NGS library preparation and provide a solution for decentralized testing that does not require specially trained personnel. With a batch size of 6, smaller hospitals and laboratories can perform NGS library preparation on fresh or archived samples with a fast turn-around-time of two days, facilitating earlier reporting to speed up diagnosis and treatment. The Helaxy NGS fluidic device performs sample nucleic acid extraction and a 2-step PCR in a plastic fluidic card, which is the sole consumable required for the extraction and library prep process.The sample is applied to the fluidic card by manual pipetting followed by loading of the card into the device. Nucleic acids are extracted with a combination of magnetic bead pull-down and centrifugal force in a purification chamber to give an eluate that is spun to a separate PCR chamber that uses the concept of thermal induction and cycling. Barcoding for library preparation follows within the same PCR chamber and the resulting product is transferred into another fluidic card for size selection purification, normalization and pooling to generate a library for input into compatible NGS sequencers, e.g., but not confined to, Illumina instruments. Results In order to demonstrate clinical utility we have shown that the Helaxy NGS fluidic device can successfully extract nucleic acid from at least three sample types: SARS-CoV-2 RNA from nasopharyngeal swabs, HIV RNA from plasma and 16S RNA from stools. Using in-house and commercial assays, we have generated libraries of target amplicons using the fluidic device workflow followed by sequencing with Illumina sequencers iSeq and MiSeq. Sequencing results analysed and reported by the Helaxy NGS Bioinformatics Reporting Pipeline revealed expected variants and mutations in the samples. We correctly identified all the unique mutations such as L211del and G446S in commercially available inactivated Omicron BA1 variant of the SARS-CoV-2 virus as well as characterised drug-resistant mutations such as L90M in the Protease gene and D67N and Y181C in the Reverse Transcriptase gene in commercially available inactivated HIV-1 virus. Conclusion The Helaxy NGS fluidic device can thus effectively provide an easy-to-use automated solution for sample library preparation in decentralized facilities to reduce the turn-around-time from sample collection to result reporting for clinical diagnosis and treatment.

Establishing molecular pathology curriculum for pathology trainees and continued medical education: a collaborative work from the Molecular Pathology Study Group of the Korean Society of Pathologists.

The importance of molecular pathology tests has increased during the last decade, and there is a great need for efficient training of molecular pathology for pathology trainees and as continued medical education. The Molecular Pathology Study Group of the Korean Society of Pathologists appointed a task force composed of experienced molecular pathologists to develop a refined educational curriculum of molecular pathology. A 3-day online educational session was held based on the newly established structure of learning objectives; the audience were asked to score their understanding of 22 selected learning objectives before and after the session to assess the effect of structured education. The structured objectives and goals of molecular pathology was established and posted as a web-based interface which can serve as a knowledge bank of molecular pathology. A total of 201 pathologists participated in the educational session. For all 22 learning objectives, the scores of self-reported understanding increased after educational session by 9.9 points on average (range, 6.6 to 17.0). The most effectively improved items were objectives from next-generation sequencing (NGS) section: 'NGS library preparation and quality control' (score increased from 51.8 to 68.8), 'NGS interpretation of variants and reference database' (score increased from 54.1 to 68.0), and 'whole genome, whole exome, and targeted gene sequencing' (score increased from 58.2 to 71.2). Qualitative responses regarding the adequacy of refined educational curriculum were collected, where favorable comments dominated. Approach toward the education of molecular pathology was refined, which would greatly benefit the future trainees.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Development and clinical applications of an enclosed automated targeted NGS library preparation system

The rapid development of next-generation sequencing (NGS) technology has promoted its wide clinical application in precision medicine for oncology. However, laborious and time-consuming manual operations, highly skilled personnel requirements, and cross-contamination are major challenges for the clinical implementation of NGS technology-based tests. The Automated NGS Diagnostic Solutions (ANDiS) 500 system is a fully enclosed cassette-dependent automated NGS library preparation system. This platform could produce qualified targeted amplicon library in three steps with only 15 min of hands-on time. Rigorous cross-contamination test using simulated contaminant plasmids confirmed that the design of disposable cassette guarantees zero sample cross-contamination. The BRCA1 and BRCA2 mutation detection panel and gastrointestinal cancer-related gene analysis panel for the ANDiS 500 platform showed 100% accuracy and precision in detecting germ-line mutations and somatic mutations respectively. Furthermore, those panels showed 100% concordance with verified methods in a prospective cohort study enrolling 363 patients and a cohort of 45 pan-cancer samples. In conclusion, the ANDiS 500 automated platform could overcome major challenges for implementing NGS assays clinically and is eligible for routine clinical tests.

FastContext: A tool for identification of adapters and other sequence patterns in next generation sequencing (NGS) data.

The development of next generation sequencing (NGS) methods has created the need for detailed analysis and control of each protocol step. NGS library preparation protocols may include steps with incorporation of various service sequences, such as sequencing adapters, primers, sample-, cell-, and molecule-specific barcodes. Despite a fairly high level of current knowledge, during the protocol development process researches often have to deal with various kinds of unexpected experiment outcomes, which result either from lack of information, lack of knowledge, or defects in reagent manufacturing. Detection and analysis of service sequences, their distribution and linkage may provide important information for protocol optimization. Here we introduce FastContext, a tool designed to analyze NGS read structure, based on sequence features found in reads, and their relative position in the read. The algorithm is able to create human readable read structures with user-specified patterns, to calculate counts and percentage of every read structure. Despite the simplicity of the algorithm, FastContext may be useful in read structure analysis and, as a result, can help better understand molecular processes that take place at different stages of NGS library preparation. The project is open-source software, distributed under GNU GPL v3, entirely written in the programming language Python, and based on well-maintained packages and commonly used data formats. Thus, it is cross-platform, may be patched or upgraded by the user if necessary. The FastContext package is available at the Python Package Index (https://pypi.org/project/FastContext), the source code is available at GitHub (https://github.com/regnveig/FastContext).

Protocol and Matters for Consideration for the Treatment of Polymerase Chain Reaction Contamination in Next-Generation Sequencing Laboratories

Abstract Objective: The contamination of polymerase chain reaction (PCR) samples in molecular diagnostic laboratories can cause serious consequences. Internal quality control efforts are often inadequate, especially in clinical next-generation sequencing (NGS) laboratories. Methods: In this study, we retrospectively investigated an incidence of PCR contamination and its decontamination process in a clinical laboratory. We performed a series of measures for decontamination. Taqman fluorescence quantification was carried out to determine the presence of contaminating DNA. SYBR-Green PCR was conducted to evaluate the effect of chlorine disinfectant on NGS library preparation. Results: Through a series of elimination measures undertaken over 8 weeks, the decontamination process was verified as reliable. Almost no contamination was detected. Chlorine disinfectant should be forbidden in Illumina NGS laboratories because it may cause the failure of library preparation. Conclusion: Our prevention and decontamination strategies could effectively eliminate PCR amplicons. Chlorine disinfectants should not be used in Illumina NGS laboratories.

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.