Library Preparation Protocol Research Articles

Isolation of High-Molecular-Weight (HMW ) DNA from Fusarium oxysporum for Long-Read Sequencing.

In many instances Fusarium oxysporum genomes are complex and challenging to assemble mainly due to the increased number of repetitive elements and variable numbers of supernumerary chromosomes, which are primarily associated with pathogenicity. Hence, to obtain the accurate F. oxysporum genome assembly and high-resolution sequence information, protocols for versatile, reliable, and high recovery of high-quality DNA for diverse sequencing platforms are instrumental. Here, we describe two protocols for the isolation of DNA from isolates of F. oxysporum. One is a quick and easy method for high-throughput extraction of DNA to rapidly screen diverse isolates by marker-assisted PCR analysis. Another is to harvest high-quality and high-molecular-weight DNA for whole-genome sequencing. In addition, we also include a library preparation protocol optimized for the third-generation sequencing technology using the portable MinION device to obtain long-read sequences. These protocols can be potentially further applied for all Fusarium spp. and other fungal pathogens, including Verticillium.

Establishment of a near-contiguous genome sequence of the citric acid producing yeast Yarrowia lipolytica DSM 3286 with resolution of rDNA clusters and telomeres.

Yarrowia lipolytica is an oleaginous yeast that is particularly suitable for the sustainable production of secondary metabolites. The genome of this yeast is characterized by its relatively large size and its high number of different rDNA clusters located in its telomeric regions. However, due to the presence of long repetitive elements in the sub-telomeric regions, rDNA clusters and telomeres are missing in current genome assemblies of Y. lipolytica. Here, we present the near-contiguous genome sequence of the biotechnologically relevant strain DSM 3286. We employed a hybrid assembly strategy combining Illumina and nanopore sequencing reads to integrate all six rDNA clusters as well as telomeric repeats into the genome sequence. By fine-tuning of DNA isolation and library preparation protocols, we were able to create ultra-long reads that not only contained multiples of mitochondrial genomes but also shed light on the inter- and intra-chromosomal diversity of rDNA cluster types. We show that there are ten different rDNA units present in this strain that additionally appear in a predefined order in a cluster. Based on single reads, we also demonstrate that the number of rDNA repeats in a specific cluster varies from cell to cell within a population.

Is amplification bias consequential in transposon sequencing (TnSeq) assays? A case study with a Staphylococcus aureus TnSeq library subjected to PCR-based and amplification-free enrichment methods.

As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq data consist of millions of nucleotide sequence reads that are generated by PCR amplification of transposon-genomic junctions. Reads mapping to the junctions are enumerated thus providing information on the number of transposon insertion mutations in each individual gene. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles, and when including a nested PCR, in the enrichment step. Additionally, we present nCATRAs - a novel, amplification-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore MinION sequencing. nCATRAs achieved 54 and 23% enrichment of the transposons and transposon-genomic junctions, respectively, over background genomic DNA. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of TnSeq insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable, but researchers interested in profiling putative essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, nCATRAs is comparable to traditional PCR-based methods (Kendall’s correlation=0.896–0.897) although the latter remain superior owing to their accessibility and high sequencing depth.

Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a <em>Burkholderia gladioli</em> Symbiont of <em>Lagria villosa</em> Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles.

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identifymolecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette iscarried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing.

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript. In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment. Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer's guidelines with some degree of optimization. The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Real-time mapping of nanopore raw signals.

MotivationOxford Nanopore Technologies sequencing devices support adaptive sequencing, in which undesired reads can be ejected from a pore in real time. This feature allows targeted sequencing aided by computational methods for mapping partial reads, rather than complex library preparation protocols. However, existing mapping methods either require a computationally expensive base-calling procedure before using aligners to map partial reads or work well only on small genomes.ResultsIn this work, we present a new streaming method that can map nanopore raw signals for real-time selective sequencing. Rather than converting read signals to bases, we propose to convert reference genomes to signals and fully operate in the signal space. Our method features a new way to index reference genomes using k-d trees, a novel seed selection strategy and a seed chaining algorithm tailored toward the current signal characteristics. We implemented the method as a tool Sigmap. Then we evaluated it on both simulated and real data and compared it to the state-of-the-art nanopore raw signal mapper Uncalled. Our results show that Sigmap yields comparable performance on mapping yeast simulated raw signals, and better mapping accuracy on mapping yeast real raw signals with a 4.4× speedup. Moreover, our method performed well on mapping raw signals to genomes of size >100 Mbp and correctly mapped 11.49% more real raw signals of green algae, which leads to a significantly higher F1-score (0.9354 versus 0.8660).Availability and implementationSigmap code is accessible at https://github.com/haowenz/sigmap.Supplementary informationSupplementary data are available at Bioinformatics online.

Abstract 2287: Detection of RNA-Seq library preparation type via random forest

Abstract Library preparation protocols for RNA sequencing (RNA-Seq) libraries vary and affect the downstream analysis of RNA-Seq data. Available RNA sequencing datasets often lack library construction protocol information in the metadata. This information is necessary to be able to compare RNA sequencing datasets appropriately. For example, non-polyadenylated transcripts are measured when a ribosomal RNA-depletion method is used (riboD library preparation), but not when transcripts are selected by poly(A) tails (polyA library preparation). Without knowing the library preparation method, it would appear that the non-polyadenylated transcripts were expressed at much higher levels in the samples prepared via riboD. In order to tackle this issue, we have developed a Random Forest classifier that can delineate between riboD and polyA RNA-Seq datasets. A grid search method was applied to the number of trees, maximum depth, and the minimum datasets included in each leaf node, which determined the best parameters to be 100, 8, and 1 respectively. However, applying the model on the Pediatric Brain Tumor Atlas (PBTA) showed strong overfitting for polyA samples. We examined the performance of the model investigating each maximum depth increment from 1 to 8, and we determined that the best performance on our validation set was achieved with maximum depth of 1. We subsequently proceeded to train our classifier on our own curated compendiums of pediatric cancer polyA and riboD samples including 188 and 264 samples respectively, after balancing the two datasets in terms of disease prevalence, and selecting the top 5,000 most variable genes as the default input dimensionality of the model. For samples whose genes do not exactly match the predetermined 5,000 genes, we substitute the expression of the missing gene with the mean expression of the gene observed in the training data. We show that it achieves 100% classification accuracy of samples to their respective library preparation protocols in GTEX (all polyA), CCLE (all polyA), and for 7 of 9 SRA projects. Five of the SRA projects contained only riboD datasets, 3 contained only all polyA datasets, and one was 50% riboD, and 50% polyA. Notably the SRA datasets are not all cancer related datasets, showing the power of our model to distinguish between library preparation protocols in vastly different settings. The model will become available on Docker so that it is readily, and easily accessible for application on new samples. Our model serves as an important step towards robust library preparation identification. Including samples in the training procedure from diverse contexts would make our model more widely applicable. Need summary statement here and/or future work statement Citation Format: Ioannis Anastopoulos, Holly Beale, Geoff Lyle, Allison Cheney, Olena M. Vaske, Joshua M. Stuart. Detection of RNA-Seq library preparation type via random forest [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2287.

Abstract 2098: Twist Fast Hybridization targeted methylation sequencing: a tunable target enrichment solution for methylation detection

Abstract DNA methylation is a genetic occurrence that plays a principal role in the epigenetic regulation of gene expression. Genome wide association studies (GWAS) have linked differential methylation patterns, especially at cytosines in CpG context, to disease states like cancer and obesity. Well-known chemical and enzymatic techniques are available to detect the methylation state of cytosines throughout the genome. With Next-Generation Sequencing (NGS) becoming increasingly affordable and high-throughput, methylation sequencing has become a popular and accessible approach for disease detection. Correspondingly, there is an increasing demand for targeted methylation sequencing techniques that enrich for CpG-rich genomic regions of interest. Such techniques have historically been inefficient due to the reduced sequence complexity caused by bisulfite or enzymatic library preparation methods. These methods involve the conversion of unmethylated cytosines to uracils, subsequently resulting in thymines after PCR amplification, and inherently increase similarities between library sequences in a given population. This effect causes issues with specificity during hybrid capture since baits and targets with repetitive sub-sequences can incidentally hybridize to non-targeted molecules during capture. In this poster, we present the Twist Targeted Methylation Sequencing Product, an end-to-end solution for preparing methyl-seq libraries and performing in-solution hybrid capture. We demonstrate benefits of using the NEBNext Enzymatic Methyl-seq Methylation Library Preparation protocol in our system, compared to traditional bisulfite treatment, and discuss approaches that were taken to maximize target enrichment specificity and uniformity. First, we introduce Twist's custom panel design approach, which leverages Twist's oligo manufacturing capabilities and uses predictive methods to remove probes most likely to cause poor capture performance. Second, we present methylation-specific Twist Fast Hybridization Target Enrichment protocol tunability, showing how FastHyb Wash Buffer 1 temperature and hybridization time can be used to manipulate performance metrics. Third, we introduce Twist's Methylation Enhancer, a novel blocker set that has shown to alleviate capture specificity issues across a wide range of methylation panels. Together, the data in this study showcase the Twist Targeted Methylation Sequencing Product as an efficient and flexible solution for custom methylation detection. Citation Format: Brenton I. Graham, Leonardo Arbiza, Kristin Butcher, Siyuan Chen, Richard Gantt, Sabina Gude, Rebecca Nugent, Christina Thompson, Ramsey Zeitoun. Twist Fast Hybridization targeted methylation sequencing: a tunable target enrichment solution for methylation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2098.

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

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

Evaluation of whole-genome DNA methylation sequencing library preparation protocols

BackgroundWith rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types.ResultsAn assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor.ResultsThe NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development.

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

BoardION: real-time monitoring of Oxford Nanopore sequencing instruments

BackgroundOne of the main advantages of the Oxford Nanopore Technology (ONT) is the possibility of real-time sequencing. This gives access to information during the experiment and allows either to control the sequencing or to stop the sequencing once the results have been obtained. However, the ONT sequencing interface is not sufficient to explore the quality of sequencing data in depth and existing quality control tools do not take full advantage of real-time data streaming.ResultsHerein, we present BoardION, an interactive web application to analyze the efficiency of ONT sequencing runs. The interactive interface of BoardION allows users to easily explore sequencing metrics and optimize the quantity and the quality of the data generated during the experiment. It also enables the comparison of multiple flowcells to assess library preparation protocols or the quality of input samples.ConclusionBoardION is dedicated to people who manage ONT sequencing instruments and allows them to remotely and in real time monitor their experiments and compare multiple sequencing runs. Source code, a Docker image and a demo version are available at http://www.genoscope.cns.fr/boardion/.

Short single‐stranded DNA with putative non‐canonical structures comprises a novel class of plasma cell‐free DNA

Cell-free DNA (cfDNA) in human blood is currently being investigated as a minimally invasive, highly informative biomarker. Besides carrying genetic variations, cfDNA can also provide useful epigenetic information through its methylation and fragmentation pattern. Previous cfDNA studies have focused on nucleosome-sized fragments, leaving shorter ones neglected. Hence, we intended to investigate whether shorter cfDNA fragments exist in the blood or not. Using an improved cfDNA purification protocol and a 3ʹ-end labeling method, we found that human plasma contains DNA fragments of approximately 50 nucleotides, present at a molar concentration largely comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by using popular cfDNA isolation methods, and they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library preparation protocol based on a ssDNA ligation technique unique developed by us and applied it to cfDNA isolated with the improved purification protocol. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

EZcount: An all-in-one software for microRNA expression quantification from NGS sequencing data

MicroRNAs (miRNAs) are short endogenous molecules of RNA that influence cell regulation by suppressing genes. Their ubiquity throughout all branches of the tree of life has suggested their central role in many cellular functions. Nowadays, several personalized medicine applications rely on miRNAs as biomarkers for diagnoses, prognoses, and prediction of drug response. The increasing ease of sequencing miRNAs contrasts with the difficulty of accurately quantifying their concentration. The use of general purpose aligners is only a partial solution as they have limited possibilities to accurately solve ambiguous mapping due to the short length of these sequences.We developed EZcount, an all-in-one software that, with a single command, performs the entire quantification process: from raw fastq files to read counts. Experiments show that EZcount is more sensitive and accurate than methods based on sequence alignment, independently of the library preparation protocol and sequencing machine. The parallel architecture of EZcount makes it fast enough to process a sample in minutes using a standard workstation.EZcount runs on all of the most common operating systems (Linux, Windows and MacOS) and is freely available for download at https://gitlab.com/BioAlgo/miR-pipe.A detailed description of the datasets, the raw experimental results, and all the scripts used for testing are available as supplementary material.

Quantitative nucleotide resolution profiling of RNA cytidine acetylation by ac4C-seq.

A prerequisite to defining the transcriptome-wide functions of RNA modifications is the ability to accurately determine their location. Here, we present N4-acetylcytidine (ac4C) sequencing (ac4C-seq), a protocol for the quantitative single-nucleotide resolution mapping of cytidine acetylation in RNA. This method exploits the kinetically facile chemical reaction of ac4C with sodium cyanoborohydride under acidic conditions to form a reduced nucleobase. RNA is then fragmented, ligated to an adapter at its 3' end and reverse transcribed to introduce a non-cognate nucleotide at reduced ac4C sites. After adapter ligation, library preparation and high-throughput sequencing, a bioinformatic pipeline enables identification of ac4C positions on the basis of the presence of C→T misincorporations in reduced samples but not in controls. Unlike antibody-based approaches, ac4C-seq identifies specific ac4C residues and reports on their level of modification. The ac4C-seq library preparation protocol can be completed in ~4 d for transcriptome-wide sequencing.

A streamlined solution for processing, elucidating and quality control of cyclobutane pyrimidine dimer sequencing data.

UV radiation may lead to melanoma and nonmelanoma skin cancers by causing helix-distorting DNA damage such as cyclobutane pyrimidine dimers (CPDs). These DNA lesions, if located in important genes and not repaired promptly, are mutagenic and may eventually result in carcinogenesis. Examining CPD formation and repair processes across the genome can shed light on the mutagenesis mechanisms associated with UV damage in relevant cancers. We recently developed CPD-Seq, a high-throughput and single-nucleotide resolution sequencing technique that can specifically capture UV-induced CPD lesions across the genome. This novel technique has been increasingly used in studies of UV damage and can be adapted to sequence other clinically relevant DNA lesions. Although the library preparation protocol has been established, a systematic protocol to analyze CPD-Seq data has not been described yet. To streamline the various general or specific analysis steps, we developed a protocol named CPDSeqer to assist researchers with CPD-Seq data processing. CPDSeqer can accommodate both a single- and multiple-sample experimental design, and it allows both genome-wide analyses and regional scrutiny (such as of suspected UV damage hotspots). The runtime of CPDSeqer scales with raw data size and takes roughly 4 h per sample with the possibility of acceleration by parallel computing. Various guiding graphics are generated to help diagnose the performance of the experiment and inform regional enrichment of CPD formation. UV damage comparison analyses are set forth in three analysis scenarios, and the resulting HTML pages report damage directional trends and statistical significance. CPDSeqer can be accessed at https://github.com/shengqh/cpdseqer .