Next-generation Sequencing Libraries Research Articles

Decoding genetic and epigenetic information embedded in cell free DNA with adapted SALP-seq.

Cell free DNA (cfDNA) in human plasma carries abundant information, which has therefore been the key sample for noninvasive prenatal testing (NIPT) and liquid biopsy. Especially by using the rapidly developed next-generation sequencing (NGS) techniques, the genetic and epigenetic information embedded in cfDNA has been effectively and extensively decoded. In this process, a key step is to construct the NGS library. Due to its high degradation, the single strand-based method was reported to be more qualified to construct the NGS library of cfDNA. In order to develop a new simple method for this application, this study adapted our recently developed single strand adaptor library preparation (SALP) method for constructing an NGS library of cfDNA. In the improved method, cfDNA was firstly denatured into single strands and then ligated with a single strand adaptor (SSA) that had a 3' overhang of 3 random bases by using T4 DNA ligase. The SSA-ligated DNA was converted into double-stranded DNA with an additional adenine at the other end by polymerizing with Taq polymerase. Next, a barcode T adaptor (BTA) was ligated to this end. Finally, the cfDNA ligated with two adaptors was amplified with the Illumina-compatible primers for NGS. Using the method, this study successfully sequenced 20 cfDNA samples from 16 esophageal cancer patients and 4 healthy people. By bioinformatics analysis, this study found the genetic and epigenetic difference between patients and healthy people and identified 23 epigenetic and 28 genetic altered esophageal cancer-specific genes.

Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies

The emergence of highly sensitive molecular diagnostic approaches, such as droplet digital PCR, has allowed the accurate identification of low-frequency variant alleles in clinical specimens; however, the multiplex capabilities of droplet digital PCR for variant detection are inadequate. The incorporation of molecular barcodes or unique IDs into next-generation sequencing libraries through PCR has enabled the detection of low-frequency variant alleles across multiple genomic regions. However, rational library preparation and sequencing data analytic strategies that integrate molecular barcodes have rarely been applied to clinical settings. In this study, we evaluated the parameters that are crucial in the use of molecular barcodes in next-generation sequencing for genotyping clinical specimens from patients with hematologic malignancies. The uniform incorporation of molecular barcodes into DNA templates through PCR was found to be crucial, and the extent of uniformity was governed by multiple interdependent variables. An error elimination strategy was developed for removing sequencing background errors by using molecular barcode sequence information as an alternative to the conventional error correction approach. This approach was successfully used to identify mutations with frequencies as low as 0.15%, and the clonal heterogeneity of hematologic malignancies was revealed. These findings have implications for elucidating heterogeneity and temporal and spatial clonal evolution, evaluating response to therapy, and monitoring relapse in patients with hematologic malignancies.

Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation

High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon.

Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.

Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody™ Single-Cell Analysis System.

In this review, we describe the BD Rhapsody™ Single-Cell Analysis System, a platform that allows high-throughput capture of nucleic acids from single cells using a simple cartridge workflow and a multitier barcoding system. The resulting captured information can be used to generate various types of next-generation sequencing (NGS) libraries, including whole transcriptome analysis for discovery biology and targeted RNA analysis for high sensitivity transcript detection. The BD Rhapsody system can be used with emerging applications, such as BD™ AbSeq assays, to profile gene expression in both mRNA and protein level to provide ultra-high resolution analysis of single cells.

NGS-Based High-Throughput Screen to Identify MicroRNAs Regulating Growth of B-Cell Lymphoma.

MicroRNAs (miRNAs) play important roles in development, differentiation, and homeostasis by regulating protein translation. In B-cell lymphoma, many miRNAs have altered expression levels, and for a limited subset of them, experimental data supports their functional relevance in lymphoma pathogenesis. This chapter describes an unbiased next-generation sequencing (NGS)-based high-throughput screening approach to identify miRNAs that are involved in the control of cell growth. First, we provide a protocol for performing high-throughput screening for miRNA inhibition and overexpression. Second, we describe the procedure for next-generation sequencing library preparation. Third, we provide a workflow for data analysis.

Whole genome sequencing of Indonesian dengue virus isolates using next-generation sequencing

Indonesia is a tropical country and hyperendemic for dengue. The disease prevalently affected Indonesian and it caused high morbidity and substantial economic burden. This vector-borne viral disease is caused by infection of dengue viruses (DENVs), which are the member of Flaviviridae family. While most of dengue studies in Indonesia focused on the epidemiology, the clinical aspects, the vectors, and to certain extent the virology, there were still gaps in the DENVs genomic aspects. Considering their high mutation rate, the DENVs were known for their high genetic diversity and it might affect the characteristics of the viruses. Comprehensive DENV genomic data were thus important for many aspects of disease management, including virus surveillance, pathogenesis, diagnostics, antiviral drug design, and vaccine development. We established in this study a method for DENV whole genome sequencing using the advanced Next-Generation Sequencing (NGS) and Nextera XT DNA library preparation kit, coupled with simplified bioinformatic analysis methods. The Indonesian DENVs from four serotypes were isolated from patients’ sera, while library was prepared from enriched templates and sequenced using Illumina NGS. Our study highlighted the potential of a robust NGS method in producing whole genome sequence of DENVs, which would be important for future dengue studies.

Enhanced bioinformatic profiling of VIDISCA libraries for virus detection and discovery

VIDISCA is a next-generation sequencing (NGS) library preparation method designed to enrich viral nucleic acids from samples before highly-multiplexed low depth sequencing. Reliable detection of known viruses and discovery of novel divergent viruses from NGS data require dedicated analysis tools that are both sensitive and accurate. Existing software was utilised to design a new bioinformatic workflow for high-throughput detection and discovery of viruses from VIDISCA data. The workflow leverages the VIDISCA library preparation molecular biology, specifically the use of Mse1 restriction enzyme which produces biological replicate library inserts from identical genomes. The workflow performs total metagenomic analysis for classification of non-viral sequence including parasites and host, and separately carries out virus specific analyses. Ribosomal RNA sequence is removed to increase downstream analysis speed and remaining reads are clustered at 100% identity. Known and novel viruses are sensitively detected via alignment to a virus-only protein database, and false positives are removed. A new cluster-profiling analysis takes advantage of the viral biological replicates produced by Mse1 digestion, using read clustering to flag the presence of short genomes at very high copy number. Importantly, this analysis ensures that highly repeated sequences are identified even if no homology is detected, as is shown here with the detection of a novel gokushovirus genome from human faecal matter. The workflow was validated using read data derived from serum and faeces samples taken from HIV-1 positive adults, and serum samples from pigs that were infected with atypical porcine pestivirus.

SMARTcleaner: identify and clean off-target signals in SMART ChIP-seq analysis

BackgroundNoises and artifacts may arise in several steps of the next-generation sequencing (NGS) process. Recently, an NGS library preparation method called SMART, or Switching Mechanism At the 5′ end of the RNA Transcript, is introduced to prepare ChIP-seq (chromatin immunoprecipitation and deep sequencing) libraries from small amount of DNA material, using the DNA SMART ChIP-seq Kit. The protocol adds Ts to the 3′ end of DNA templates, which is subsequently recognized and used by SMART poly(dA) primers for reverse transcription and then addition of PCR primers and sequencing adapters. The poly(dA) primers, however, can anneal to poly(T) sequences in a genome and amplify DNA fragments that are not enriched in the immunoprecipitated DNA templates. This off-target amplification results in false signals in the ChIP-seq data.ResultsHere, we show that the off-target ChIP-seq reads derived from false amplification of poly(T/A) genomic sequences have unique and strand-specific features. Accordingly, we develop a tool (called “SMARTcleaner”) that can exploit these features to remove SMART ChIP-seq artifacts. Application of SMARTcleaner to several SMART ChIP-seq datasets demonstrates that it can remove reads from off-target amplification effectively, leading to significantly improved ChIP-seq peaks and results.ConclusionsSMARTcleaner could identify and clean the false signals in SMART-based ChIP-seq libraries, leading to improvement in peak calling, and downstream data analysis and interpretation.

Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia

Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy.Methodology:A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing.Results:179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger.Conclusions:Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4).Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples.None of the single mutation patients had presented in a blast crisis or an accelerated phase. DisclosuresNo relevant conflicts of interest to declare.

An Improved NGS Library Construction Approach Using DNA Isolated from Human Cancer Formalin-Fixed Paraffin-Embedded Samples.

Identification of genomic alterations from formalin-fixed paraffin-embedded (FFPE) samples using next-generation sequencing (NGS) is very important for cancer-targeted therapy today. To achieve a higher efficiency and shorter turn-around time for NGS library preparation, here, we compared NGS library preparation processes and outcomes with three commercial library construction methods and two hybridization capture methods thus, developed an improved NGS library construction approach. This improved approach took advantage of both methods and resulted in a higher output from the same input DNA, including higher library construction success rate, higher probe capture rate, and shorter turn-around time. Using this approach, targeted region libraries could be constructed within only 1 day for FFPE samples; therefore, this approach has potential applications of NGS in routine clinical tests. Anat Rec, 302:941-946, 2019. © 2018 Wiley Periodicals, Inc.

Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.

The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at https://doi.org/www.hi-tom.net/hi-tom/ ), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.

BRCA-analyzer: Automatic workflow for processing NGS reads of BRCA1 and BRCA2 genes

The use of targeted next-generation sequencing (NGS) provides great new opportunities for molecular and medical genetics. However, in order to take advantage of these opportunities, we need to have reliable tools for extracting the necessary information from the huge amount of data generated by NGS. Here we present our automatic multithreaded workflow for processing NGS data of BRCA1 and BRCA2 genes obtained with NGS technology named BRCA-analyzer. Optimizing it on the sequencing data of 899 samples from 693 patients, we were able to find the most reliable tools and adjust their parameters in such a way that all pathogenic variants found were confirmed by Sanger’s sequencing. For 82 and 24 DNA samples from blood and formalin-fixed paraffin-embedded blocks, NGS libraries were prepared with GeneRead BRCA panel v2 (Qiagen). The reads obtained were processed with BRCA-analyzer and Qiagen GeneRead Data analysis workflow. In total 27 pathogenic variants were found and confirmed by Sanger’s sequencing, with all of them determined with BRCA-analyzer. Qiagen GeneRead Data analysis discarded 5 true pathogenic variants due to their location in homopolymeric sequence stretches. For other 793 samples, libraries were prepared by the in-house method, and NGS data were analyzed by BRCA-analyzer in comparison to another free automatic amplicon NGS workflow Canary. From total 137 pathogenic variations, BRCA-analyzer found 135 and Canary 123. Mutations were missed by BRCA-analyzer due to the trimming primer sequences from reads before mapping to be fixed in the next version. On the freely available NGS data, we showed that BRCA-analyzer could also be used for hybrid capture gene panels, although it needs more extensive testing on such library preparation methods. Thus, BRCA-analyzer is an automatic workflow for processing NGS data of BRCA1/2 genes with variant filters adapted to amplicon-based targeted NGS data. BRCA-analyzer can be used to identify germline as well as somatic mutations. BRCA-analyzer is freely available at https://github.com/aakechin/BRCA-analyzer.

Universal Target Capture of HIV Sequences From NGS Libraries

Background: Global surveillance of viral sequence diversity is needed to keep pace with the constant evolution of HIV. Recent next generation sequencing (NGS) methods have realized the goal of sequencing circulating virus directly from patient specimens. Yet, a simple, universal approach that maximizes sensitivity and sequencing capacity remains elusive. Here we present a novel HIV enrichment strategy to yield near complete genomes from low viral load specimens.Methodology: A non-redundant biotin-labeled probe set (HIV-xGen; n = 652) was synthesized to tile all HIV-1 (groups M, N, O, and P) and HIV-2 (A and B) strains. Illumina Nextera barcoded libraries of either gene-specific or randomly primed cDNA derived from infected plasma were hybridized to probes in a single pool and unbound sequences were washed away. Captured viral cDNA was amplified by Illumina adaptor primers, sequenced on a MiSeq, and NGS reads were demultiplexed for alignment with CLC Bio software.Results: HIV-xGen probes selectively captured and amplified reads spanning the entirety of the HIV phylogenetic tree. HIV sequences clearly present in unenriched libraries of specimens but previously not observed due to high host background levels, insufficient sequencing depth or the extent of multiplexing, were now enriched by >1,000-fold. Thus, xGen selection not only substantially increased the depth of existing sequence, but also extended overall genome coverage by an average of 40%. We characterized 50 new, diverse HIV strains from clinical specimens and demonstrated a viral load cutoff of approximately log 3.5 copies/ml for full length coverage. Genome coverage was <20% for 5/10 samples with viral loads <log 3.5 copies/ml and >90% for 35/40 samples with higher viral loads.Conclusions: Characterization of >20 complete genomes at a time is now possible from a single probe hybridization and MiSeq run. With the versatility to capture all HIV strains and the sensitivity to detect low titer specimens, HIV-xGen will serve as an important tool for monitoring HIV sequence diversity.

Performance comparison of blood collection tubes as liquid biopsy storage system for minimizing cfDNA contamination from genomic DNA.

The ratio of target cfDNA in total plasma is low. The abundant gDNA background resulting from blood cell lysis caused by improper operation has become a major obstacle to accurately measure cfDNA. In this study, we investigated the storage capacity of three blood cell collection tubes (BCTs) in the prevention of genomic DNA contamination caused by white blood cell rupture and evaluated their performance when they were utilized combining with highly sensitive mutation detection technology. Blood samples were drawn from six healthy blood donors and stored in three types of BCTs (BD K2 -EDTA tube, Roche tube, and Streck tube). Plasma samples were isolated at specific time points (day 0, day 3, day 7, and day 14) for content analysis. Roche BCT was more capable for preventing cfDNA contamination caused by white blood cell disruption within 14days, comparing with BD K2 -EDTA tube and Streck tube. Severe white blood cell lysis and gDNA contamination were found in the BD tube. The impacts of Roche and Streck tubes on the quantity and complexity of next-generation sequencing (NGS) libraries did not differ significantly within 3days, satisfying most of our daily demands. In addition, the rupture of WBC was not synchronized with hemolysis in BCTs. This study showed that capacities of blood collection tubes differed considerably in preservation of blood samples. Therefore, suitable blood collection devices should be selected to minimize gDNA contamination and to standardize blood samples processing for achieving more accurate and reliable clinical analysis of cfDNA.

Development of High-Throughput Clinical Testing ofRPGRORF15 Using a Large Inherited Retinal Dystrophy Cohort

Mutations in the ORF15 region of RPGR account for approximately half of all X-linked retinitis pigmentosa cases. However, a robust high-throughput method for the detection of ORF15 mutations has yet to be validated. We set out to develop the first clinically validated next-generation sequencing (NGS) method for the detection of mutations in this difficult-to-sequence region, including test accuracy and coverage data. As part of a blind-test, 145 research samples, previously tested by Sanger sequencing, and 81 clinical samples were evaluated using NGS of long-range PCR products fragmented with Illumina's Nextera library preparation kit (method 1), or with Centrillion's OneTube technology, supplemented with duplication analysis using an ORF15-specific in-silico array (method 2). DNA fragments were analyzed using Agilent's DNA 1000 assay, and sequencing was done on Illumina's MiSeq 2×150 or HiSeq2500 2×100. NextGENe by SoftGenetics was used for data analysis and variant calling. The Nextera library preparation method produced 24 cases of discordance due to (in order of decreasing occurrence) false-negatives, incorrectly called variants, and a false-positive. Subsequent use of a new, OneTube NGS library preparation method, supplemented with duplication analyses, resolved discordance between Sanger and NGS data in all cases. This improvement in variant detection accuracy was largely attributed to improvement in random fragmentation offered by the enzymatic OneTube method, resulting in more complete coverage of the highly repetitive ORF15 region. Minimum coverage was roughly 320 reads for Nextera and 6800 reads for OneTube (normalized for total read counts). This paper documents the first clinically validated NGS method for reliable, high-throughput sequencing of RPGR ORF15. Sensitivity and specificity of the new method were 100%, with the caveat of unclear zygosity calling for one large duplication case. These findings demonstrate a reliable and practical implementation for NGS-based diagnosis of RPGR ORF15 mutations. They also provide the foundation for targeted, high-coverage sequencing of any other repetitive regions within the genome.

Biotinylated amplicon sequencing: A method for preserving DNA samples of limited quantity

BackgroundGenomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA. Design and methodsBiotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel. ResultsRecovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications. ConclusionsWe demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.

Molecular cloning of novel transcripts of the adaptor-related protein complex 2 alpha 1 subunit (AP2A1) gene, using Next-Generation Sequencing

The adaptor-related protein (AP) complexes play important roles in cargo selection and vesicle formation, and hence in intracellular membrane trafficking. Five different AP complexes are currently known, each consisting of four subunits, known as adaptins. AP-2, the most thoroughly characterized of the five AP complexes, facilitates clathrin-mediated endocytosis. In this study, we describe the discovery and molecular cloning of seventy-seven novel alternatively spliced transcripts of the human AP2A1 gene, which encodes the αA adaptin of the AP-2 complex. For this purpose, we have used Next-Generation Sequencing (NGS), a powerful tool for studying alternative splicing. In brief, we subcultured fifty-five established human cell lines, originating from several distinct cancerous and normal tissues, extracted total RNA, and synthesized first-strand cDNA. Next, we used nested touchdown PCR to amplify the whole coding region of the AP2A1 transcripts of each cell line, mixed all PCR products, and proceeded to NGS library construction, template preparation, and semiconductor sequencing. Extensive bioinformatic analysis revealed thirteen novel splice junctions of previously annotated exons, as also verified via nested PCR with primers targeting these splice junctions. Moreover, consecutive nested PCRs led to the determination of the primary structure of seventy-seven novel AP2A1 transcripts, all of which were shown to comprise at least one premature translation termination codon, thus representing nonsense-mediated mRNA decay (NMD) candidates. NMD is a mechanism that cells use to control gene expression. Consequently, alterations in the levels of these potentially non-coding AP2A1 transcripts could lead to a decrease in the number of AP2A1 mRNA molecules, when needed. Undoubtedly, the exact role of these new APA1 splice variants merits elucidation.

Transcriptional analysis of viral mRNAs reveals common transcription patterns in cells infected by five different filoviruses.

Filoviruses are notorious viral pathogens responsible for high-consequence diseases in humans and non-human primates. Transcription of filovirus mRNA shares several common features with transcription in other non-segmented negative-strand viruses, including differential expression of genes located across the viral genome. Transcriptional patterns of Ebola virus (EBOV) and Marburg virus (MARV) have been previously described using traditional, laborious methods, such as northern blots and in vivo labeling of viral mRNAs. More recently, however, the availability of the next generation sequencing (NGS) technology has offered a more straightforward approach to assess transcriptional patterns. In this report, we analyzed the transcription patterns of four ebolaviruses—EBOV, Sudan (SUDV), Bundibugyo (BDBV), and Reston (RESTV) viruses—in two different cell lines using standard NGS library preparation and sequencing protocols. In agreement with previous reports mainly focused on EBOV and MARV, the remaining filoviruses used in this study also showed a consistent transcription pattern, with only minor variations between the different viruses. We have also analyzed the proportions of the three mRNAs transcribed from the GP gene, which are characteristic of the genus Ebolavirus and encode the glycoprotein (GP), the soluble GP (sGP), and the small soluble GP (ssGP). In addition, we used NGS methodology to analyze the transcription pattern of two previously described recombinant MARV. This analysis allowed us to correct our construction design, and to make an improved version of the original MARV expressing reporter genes.

ATAC2GRN: optimized ATAC-seq and DNase1-seq pipelines for rapid and accurate genome regulatory network inference

BackgroundChromatin accessibility profiling assays such as ATAC-seq and DNase1-seq offer the opportunity to rapidly characterize the regulatory state of the genome at a single nucleotide resolution. Optimization of molecular protocols has enabled the molecular biologist to produce next-generation sequencing libraries in several hours, leaving the analysis of sequencing data as the primary obstacle to wide-scale deployment of accessibility profiling assays. To address this obstacle we have developed an optimized and efficient pipeline for the analysis of ATAC-seq and DNase1-seq data.ResultsWe executed a multi-dimensional grid-search on the NIH Biowulf supercomputing cluster to assess the impact of parameter selection on biological reproducibility and ChIP-seq recovery by analyzing 4560 pipeline configurations. Our analysis improved ChIP-seq recovery by 15% for ATAC-seq and 3% for DNase1-seq and determined that PCR duplicate removal improves biological reproducibility by 36% without significant costs in footprinting transcription factors. Our analyses of down sampled reads identified a point of diminishing returns for increased library sequencing depth, with 95% of the ChIP-seq data of a 200 million read footprinting library recovered by 160 million reads.ConclusionsWe present optimized ATAC-seq and DNase-seq pipelines in both Snakemake and bash formats as well as optimal sequencing depths for ATAC-seq and DNase-seq projects. The optimized ATAC-seq and DNase1-seq analysis pipelines, parameters, and ground-truth ChIP-seq datasets have been made available for deployment and future algorithmic profiling.