Next-generation Sequencing Libraries Research Articles

P-725 Role of Non-Invasive Preimplantation Genetic Testing- Aneuploidy (NIPGT-A) using spent culture media (SCM) and its concordance with Trophoectoderm (TE) biopsy: A Prospective Cohort Study

Abstract Study question What is the ploidy concordance rate between spent culture media (SCM) and trophectoderm biopsy (TE) using Next Generation Sequencing (NGS) and correlation with clinical outcome? Summary answer DNA could be isolated from SCM with successful NGS library sequencing. Ploidy and per-chromosome concordance rate of TE biopsy vs SCM was 68.38% and 85.13%. What is known already Many challenges are associated with TE biopsy, and the possibility of NiPGT-A using cell free DNA (cfDNA) from SCM is very intriguing, as it will be simpler, safer and cheaper alternative to the gold standard TE biopsy. The bottlenecks associated with TE biopsy like additional equipment and trained embryologist, can be substantially reduced. The disparities in reported results in various studies comparing SCM vs TE, could be attributed to culture media contamination (maternal, paternal or environment), different laboratory workflow methodologies used for embryo culture, different whole genome amplification methods, library sequencing methods and different algorithms for analysis. Study design, size, duration Prospective cohort study was conducted in tertiary care centre from August 2021-June 2022. Ethical Approval was taken from Institute Ethics Committee. Couples with female age ≥ 35 years, one or more implantation failures, male factor infertility requiring ICSI, opting for elective single euploid blastocyst transfer, were included in study. Forty four blastocysts were obtained from 14 patients who underwent IVF/ICSI cycles, gave consent for TE biopsy and SCM collection for PGT-A and NiPGT-A respectively. Participants/materials, setting, methods Individualised ovarian stimulation was carried out with GnRH antagonist protocol. ICSI was done for fertilization. Embryos were cultured in sequential medium. On day 3, after thorough washing embryo was cultured separately in 10μl micro-droplets. SCM was collected on day-5, before biopsy. DNA extraction, amplification and library preparation from TE biopsy and SCM was done using Veriseq PGS kit (Illumina) and sequencing was done on Illumina MiSeq system. Euploid embryo transfer was done in FET cycle Main results and the role of chance Out of 44 blastocysts, 31 were day 5 and 13 were day 6. WGA-DNA from TE biopsy and SCM was 100% successful. Average concentration of amplified DNA obtained from TE biopsy and SCM was 34.88 ± 9.56 ng/µl, 32.3 ± 6.84 ng/µl respectively. Quality control parameters for library preparation and sequencing for samples were as per recommended standards. CNV visualization and analysis was done using BlueFuse Multi-Software (Illumina). Ploidy concordance rate could be analysed in 26 embryos, which was 68.38%, per chromosome concordance was 85.13% and sex-chromosome concordance was 73.0%. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of NiPGT-A was 66.6%, 60%, 87.5%, 30% and 65.38% respectively. On comparing day 5 and day 6 blastocysts, day 6 had better concordance rate 72.7% vs 60%, p > 0.05. On comparing good (>BB) and poor morphology (<BB)embryos, <BB had better concordance rate 83.33% vs 50%, p = 0.16. Per chromosome concordance rate was higher for <BB embryos, 90.5% vs 80.5%,p= 0.001. Full maternal cell contamination was suspected when TE was aneuploid/mosaic, XY and SCM was euploid, XX (n = 4/14), assessed only in XY embryos. Single euploid blastocyst transfer had 66.6%clinical pregnancy rate. One resulted in healthy live-birth, two on-going pregnancies, 30weeks and 12weeks period of gestation. Limitations, reasons for caution Before considering NiPGT-A in routine clinical practice, each embryology lab needs standardization, to implement necessary modifications in routine embryology protocol. Labs should ensure that embryo viability is not affected and should try to minimize chances of maternal or external contamination in SCM to reliably predict concordance rates with high confidence. Wider implications of the findings NiPGT-A is useful technique to assess ploidy but presently, NiPGT-A in combination with PGT-A, can help in prioritizing embryos according to their implantation potential. Further studies are required to find out whether NiPGT-A may serve as an alternative to PGT-A and implemented alone in routine clinical practice in future. Trial registration number CTRI/2021/04/033121

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

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

Simultaneous Analysis of Bacterial and Fungal Communities in Oral Samples from Intubated Patients in Intensive Care Unit.

Intubated patients in intensive care units (ICUs) too frequently contract ventilator-associated pneumonia or Candida infections. Oropharyngeal microbes are believed to play an important etiologic role. This study was undertaken to determine whether next-generation sequencing (NGS) can be used to simultaneously analyze bacterial and fungal communities. Buccal samples were collected from intubated ICU patients. Primers targeting the V1-V2 region of bacterial 16S rRNA and the internal transcribed spacer 2 (ITS2) region of fungal 18S rRNA were used. V1-V2, ITS2, or mixed V1-V2/ITS2 primers were used to prepare an NGS library. Bacterial and fungal relative abundances were comparable for V1-V2, ITS2, or mixed V1-V2/ITS2 primers, respectively. A standard microbial community was used to adjust the relative abundances to theoretical abundance, and NGS and RT-PCR-adjusted relative abundances showed a high correlation. Using mixed V1-V2/ITS2 primers, bacterial and fungal abundances were simultaneously determined. The constructed microbiome network revealed novel interkingdom and intrakingdom interactions, and the simultaneous detection of bacterial and fungal communities using mixed V1-V2/ITS2 primers enabled analysis across two kingdoms. This study provides a novel approach to simultaneously determining bacterial and fungal communities using mixed V1-V2/ITS2 primers.

Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

Abstract 263: Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System

Abstract This study investigates a custom 2Mb xGen Hyb panel designed against mutated gene targets implicated in several cancers using an end-to-end assay workflow on the Biomek NGeniuS platform. Automation in laboratories is advantageous to generate reproducible next generation sequencing (NGS) sample libraries, however, assay automation differs from instrument vendor to vendor but also from lab to lab with different deck layouts of the same instrument. These differences require a significant amount of optimization to design the workflow and begin generating NGS libraries. In addition to this optimization, some research, e.g., cancer research utilizes low input and/or degraded samples such as cfDNA from plasma or FFPE biopsies. Integrated DNA Technologies (IDT) and Beckman Coulter - Life Sciences together provide a walk away NGS automation solution for these research samples directly upon instrument installation with no optimization required by combining Beckman Coulter’s Biomek NGeniuS with IDT’s xGen cfDNA & FFPE DNA Library Prep kit and xGen Hybridization Capture. This trio provides a solution for laboratories to minimize technician hands-on time, error reduction, and rework while improving flexibility. IDT’s xGen cfDNA & FFPE DNA Library Prep kit utilizes novel chemistry to maximize sample input conversion, suppress adapter-dimer formation, and facilitate consensus analysis. IDT’s xGen Hybridization Capture products maintain high library diversity, obtain high on-target, and provide consistent and uniform sequencing coverage regardless of panel size. We demonstrate performance of this complete workflow with a custom 2Mb xGen Hyb panel yielding high library complexity, high on-target coverage, and capture uniformity. The combination of IDT’s xGen library preparation solutions on the NGeniuS Biomek platform provides a reliable and consistent solution that can be instantly downloaded from the portal straight to analysis of precious low input and degraded samples. Citation Format: Nicole Roseman, Laura Tucker, Francesco Criscuolo, Lynette Lewis, Li Liu, Tyler Buit, Jasmeen Mandair, Zachary Smith, Calvin Cortes. Automating next generation sequencing workflows for custom hyb panels: high quality library preparation and target enrichment on the Biomek NGeniuS System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 263.

MitoGeneExtractor: Efficient extraction of mitochondrial genes from next‐generation sequencing libraries

Abstract Mitochondrial DNA (mtDNA) sequences are often found as byproducts in next‐generation sequencing (NGS) datasets that were originally created to capture genomic or transcriptomic information of an organism. These mtDNA sequences are often discarded, wasting this valuable sequencing information. We developed MitoGeneExtractor, an innovative tool which allows to extract mitochondrial protein coding genes (PCGs) of interest from NGS libraries through multiple sequence alignments of sequencing reads to amino acid references. General references, for example on order level are sufficient for mining mitochondrial PCGs. In a case study, we applied MitoGeneExtractor to recently published genomic datasets of 1993 birds and were able to extract complete or nearly complete sequences for all 13 mitochondrial PCGs for a large proportion of libraries. Compared to an existing assembly guided sequence reconstruction algorithm, MitoGeneExtractor was faster and substantially more sensitive. We compared COI sequences mined with MitoGeneExtractor to COI databases. Mined sequences show a high sequence similarity and correct taxonomic assignment between the recovered sequence and the assigned morphospecies in most samples. In some cases of incongruent taxonomic assignments, we found evidence for contamination in NGS libraries. MitoGeneExtractor allows a fast extraction of mitochondrial PCGs from a wide range of NGS datasets. We recommend to routinely harvest and curate mitochondrial sequence information from genomic resources. MitoGeneExtractor output can be used to identify contaminated NGS libraries and to validate the species identity of the sequenced animal based on the extracted COI sequences.

A new and improved method of library preparation for non-invasive prenatal testing: plasma to library express technology.

This study aims to develop a novel library preparation method, plasma to library express technology (PLET), to construct next-generation sequencing (NGS) libraries directly from plasma without cell-free DNA (cfDNA) isolation. Peripheral blood samples (600) were obtained from a retrospective cohort of 300 pregnant women prior to invasive diagnostic testing. The samples were subsequently distributed between library preparation methodologies, with 300 samples prepared by PLET and 300 by conventional methods for non-invasive prenatal testing (NIPT) to screen for common trisomies using low-pass whole genome next generation sequencing. NIPT conducted on PLET libraries demonstrated comparable metrics to libraries prepared using conventional methods, including 100% sensitivity and specificity. Our study demonstrates the potential utility of PLET in the clinical setting and highlights its significant advantages, including dramatically reduced process complexity and markedly decreased turnaround time.

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).

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.

Sample Preparation and Differential Gene Expression Analysis of Human Cancer Cell Lines by RNA Sequencing.

RNA sequencing (RNA-seq) is a method used for the high-throughput quantification of mRNA in a biological sample. It is widely used to investigate differential gene expression between drug-resistant and sensitive cancers to identify genetic mediators of drug resistance. Here, we describe a comprehensive experimental and bioinformatic pipeline to isolate mRNA from human cell lines, prepare mRNA libraries for next-generation sequencing, and perform post-sequencing bioinformatics analyses.

Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions.

Yeast two-hybrid is a powerful approach to discover new protein-protein interactions. Traditional methods involve screening a target protein against a cDNA expression library and assaying individual positive colonies to identify interacting partners. Here we describe a simple approach to perform yeast two-hybrid screens of a cDNA expression library in batch liquid culture. Positive yeast cell populations are enriched under selection and then harvested en masse. Prey cDNAs are amplified and used as input for next-generation sequencing libraries for identification, quantification, and ranking.

Fatal outbreak among camels in Kazakhstan associated with Paeniclostridium sordellii

An outbreak of a bacterial disease with lethal cases was registered in camels in Kazakhstan in 2018. The disease was characterised by dry, harsh hacking cough and nasal discharge with froathy and bloody liquid. Camels died in a state of hyperexcitement and asphyxia. Investigation of the case has identified the pathogenic form of Paeniclostridium sordellii as the main cause of mortality in camels. The purpose of the study is to identify the cause of mortality among camels, bacteriological and molecular genetic characteristics of the pathogen. The methodological basis of the work is collection of biological samples from camels, isolating the pathogen on media, extracting nucleic acids, obtaining libraries for next generation sequencing, bioinformatic analysis of the obtained data, pathogenicity test on mice. This article describes the cultural, genetic and phenotypic characteristics of the isolated Shetpe PS18-01 strain. It was shown that the pathogenesis was mainly characterised by respiratory signs, in contrast to the most common gastrointestinal ones.

Profiling of mitochondrial heteroplasmy in single human oocytes by next-generation sequencing.

Mitochondrial DNA (mtDNA) plays a crucial role in the development of a competent oocyte. Indeed, mtDNA alterations may predispose to chromosome nondisjunction, resulting in infertility due to a reduced vitality and quality of oocytes and embryos. In this methods paper, the multiple displacement amplification approach was applied in combination with next-generation sequencing (NGS) to amplify and sequence, in single-end, the entire mtDNA of single human oocytes to directly construct genomic NGS libraries, and subsequently, to highlight and quantify the mutations they presented. The bioinformatic workflow was carried out with a specific ad hoc developed in-house software. This approach proved to be sensitive and specific, also highlighting the mutations present in heteroplasmy, showing deletion, insertion or substitution mutations in the genes involved in the respiratory chain, even if the found variants were benign or of uncertain meaning. The analysis of mtDNA mutations in the oocyte could provide a better understanding of specific genetic abnormalities and of their possible effect on oocyte developmental competence. This study shows how this approach, based on a massive parallel sequencing of clonally amplified DNA molecules, allows to sequence the entire mitochondrial genome of single oocytes in a short time and with a single analytical run and to verify mtDNA mutations.

The Landscape of Small Nucleolar RNA Expression in Multiple Myeloma

Background Small nucleolar RNAs (snoRNAs) are non-coding RNAs involved in the maturation of ribosome RNAs and various other biological processes. Emerging evidence demonstrates that snoRNAs play an essential role in tumor initiation and progression. However, their role in multiple myeloma (MM) pathogenesis remains poorly characterized. To date, the profiling of small RNAs in MM has mainly focused on microRNAs and approaches are confined to PCR- or array-based techniques. Here, we used next generation sequencing (NGS) to explore the snoRNA expression patterns of MM patients and to identify the association between snoRNAs and mRNAs, molecular subgroups of MM and clinical parameters. Methods Purified CD138+ cells from 71 newly diagnosed MMs and 4 refractory/relapsed MMs were included. Three CD19+ B cell samples from healthy donors were used as control. Total RNA was isolated with the AllPrep DNA/RNA kit (Qiagen, Germany). The mRNA and snoRNA NGS libraries were prepared using the Illumina TruSeq stranded mRNA and the NEBNext multiplex small RNA library kit, respectively. Sequencing of mRNA and snoRNA was performed on an Illumina NextSeq and NovaSeq 6000 PE 100 S1 platform, respectively. Paired-end RNA-seq reads were mapped to the hg19 reference genome using STAR. Gene expressions were quantified using featureCounts. Differentially expressed mRNAs and snoRNAs were identified using DESeq2. The Kaplan-Meier method was used for survival analyses. Progression-free survival (PFS) time was measured from enrollment to relapse or death from any cause, whichever occurred first. Results Using NGS, we observed expression of 625 full-length snoRNAs. Compared to normal B cells high levels of snoRNA expression were detected in MM plasma cells. Looking for associations between snoRNA expression and molecular subgroups of MM, we found an upregulation of 50 snoRNAs in patients with a hyperdiploid karyotype. The pattern of upregulation supported a regulatory mechanism in this molecular subgroup that goes beyond a pure copy-number effect. Even in patients with multiple trisomies the overwhelming majority (82%) of upregulated snoRNAs were located on chromosome 15, including the SNORD115 family on 15q11. In patients with gain of 1q21, all snoRNAs located on 1q25 were upregulated and the highest expression level of those snoRNAs appeared in patients with amp1q, indicating a copy-number effect. SNORD78, which was recently shown to impact outcome in prostate cancer and lung cancer, was significantly overexpressed in gain1q patients and defined a subgroup with inferior PFS (p =0.019) and overall survival (p = 0.011). SCARNA22, which was upregulated in patients with a t(4;14), was another snoRNA with a negative impact on PFS (31.06 months vs unreached, p = 0.007). To further characterize MM with upregulation of prognostically relevant snoRNAs, we analyzed bulk RNAseq of CD138-enriched MM cells and performed a pathway enrichment analysis. Comparing SNORD78 high expression group with the low group, we found 537 upregulated genes, e.g.SH2D2A, and CDK1, and 229 downregulated genes. Differentially expressed genes were enriched for cell cycle and chromosome segregation. For SCARNA22, we found 963 up- and 219 down-regulated genes. These showed enrichment for cell adhesion (upregulated in SCARNA22high) and protein targeting to the membrane or the endoplasmic reticulum (downregulated in SCARNA22high). Conclusion In this study we provide first insights into the expression patterns of snoRNAs in MM by deep sequencing. We identified snoRNAs dysregulated in MM subgroups, with two of them being associated with poor outcome. This study also shows that expression of particular snoRNAs is associated with specific chromosome gains, which is a common element in MM. Disclosures No relevant conflicts of interest to declare.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants.

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

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.

Chromatin Immunoprecipitation Sequencing (ChIP-seq) for Detecting Histone Modifications and Modifiers.

Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is the most widely used method for analyzing genome-wide DNA-protein interactions. Because there is considerable variation in the modes and strengths of DNA-protein interactions, chromatin immunoprecipitation (ChIP) protocols have been diversified and optimized for different needs. Here, we describe protocols for detecting histone modifications and modifiers using various crosslinking and immunoprecipitation conditions. We provide a complete ChIP-seq workflow covering sample preparation, immunoprecipitation, next-generation sequencing (NGS) library preparation, and data analyses.

COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens.

With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.

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.