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Whole genome sequencing (WGS) has become a gold standard for diagnosing genomic variation. Peripheral blood is a common sample source for the extraction of nucleic acids for Next-Generation Sequencing (NGS) applications. Here, we present an integrated and fully automated device design that uses new concepts of fluid mechanics, heat-mass transfer, and thermodynamics of enzymatic reactions to extract nucleic acids from the blood and perform DNA library preparation from a pre-filled plate. We demonstrate that the presented device effectively extracts dsDNA with an average of 25.03 µg/mL and 25.91 µg/mL yield from citrate-stabilized human peripheral blood stored in Fresh (4 °C) and Frozen (-20 °C) conditions, respectively. Furthermore, our method automatically extracts nucleic acids and creates a high-quality sequence-ready DNA library from blood stabilized with citrate and EDTA for 8 samples simultaneously in a single run with a total operation time of ~ 7 h. Our results show the required coverage and depth of the genome, highlighting an essential application of this device in processing blood samples for genome sequencing. [ABSTRACT FROM AUTHOR]

With the development of modern molecular biotechnology and the continuous deepening of research on gene function, Tn5 transposase has received more and more attention due to its characteristics of randomness and high efficiency in transposition. It has now been widely used as an important tool in the field of molecular biology and genetic engineering. Known for its "cut-and-paste" mechanism that allows for the integration or mobilization of DNA sequences within genomes, making it good at library construction of high-throughput sequencing. However, the weakness still exists in Tn5 transposase that hinders its further application. This article primarily reviews the architecture, catalytic mechanism, and functional modification of Tn5 transposase, as well as its application in advanced transgenic editing and library construction of sequencing. Furthermore, it provides prospects for future research in this field. [ABSTRACT FROM AUTHOR]

Shotgun metagenomics is an increasingly cost-effective approach for profiling environmental and host-associated microbial communities. However, due to the complexity of both microbiomes and the molecular techniques required to analyze them, the reliability and representativeness of the results are contingent upon the field, laboratory, and bioinformatic procedures employed. Here, we consider 15 field and laboratory issues that critically impact downstream bioinformatic and statistical data processing, as well as result interpretation, in bacterial shotgun metagenomic studies. The issues we consider encompass intrinsic properties of samples, study design, and laboratory-processing strategies. We identify the links of field and laboratory steps with downstream analytical procedures, explain the means for detecting potential pitfalls, and propose mitigation measures to overcome or minimize their impact in metagenomic studies. We anticipate that our guidelines will assist data scientists in appropriately processing and interpreting their data, while aiding field and laboratory researchers to implement strategies for improving the quality of the generated results. [ABSTRACT FROM AUTHOR]

Abstract Whole genome sequencing (WGS) has become a gold standard for diagnosing genomic variation. Peripheral blood is a common sample source for the extraction of nucleic acids for Next-Generation Sequencing (NGS) applications. Here, we present an integrated and fully automated device design that uses new concepts of fluid mechanics, heat-mass transfer, and thermodynamics of enzymatic reactions to extract nucleic acids from the blood and perform DNA library preparation from a pre-filled plate. We demonstrate that the presented device effectively extracts dsDNA with an average of 25.03 µg/mL and 25.91 µg/mL yield from citrate-stabilized human peripheral blood stored in Fresh (4 °C) and Frozen (-20 °C) conditions, respectively. Furthermore, our method automatically extracts nucleic acids and creates a high-quality sequence-ready DNA library from blood stabilized with citrate and EDTA for 8 samples simultaneously in a single run with a total operation time of ~ 7 h. Our results show the required coverage and depth of the genome, highlighting an essential application of this device in processing blood samples for genome sequencing. Graphical Abstract

Abstract Background Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. Results Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. Conclusions Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.

Background: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. Results: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. Conclusions: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy. [ABSTRACT FROM AUTHOR]

Today, the world of science is constantly challenged with new genomics, which in turn is responsible for new disease-causing targets. Hence, there is a need for developing drugs acting against such targets. Computational methods are proving to be a mainstay in the drug discovery process, mainly through virtual screening. This review discusses about the recent advancements in structure-based drug design with reference to Virtual Screening along with its procedures from ligand preparation and protein preparation, docking, scoring function, databases, and virtual (VS) algorithms. Application of Structure-based VS in combination with other virtual screening techniques has also been highlighted in this review. [ABSTRACT FROM AUTHOR]

We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications. [ABSTRACT FROM AUTHOR]

Abstract Objective Metatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (

Summary: We present an easy‐to‐reproduce manual miniaturized full‐length RNA sequencing (RNAseq) library preparation workflow that does not require the upfront investment in expensive lab equipment or long setup times. With minimal adjustments to an established commercial protocol, we were able to manually miniaturize the RNAseq library preparation by a factor of up to 1:8. This led to cost savings for miniaturized library preparation of up to 86.1% compared to the gold standard. The resulting data were the basis of a rigorous quality control analysis that inspected: sequencing quality metrics, gene body coverage, raw read duplications, alignment statistics, read pair duplications, detected transcripts and sequence variants. We also included a deep dive data analysis identifying rRNA contamination and suggested ways to circumvent these. In the end, we could not find any indication of biases or inaccuracies caused by the RNAseq library miniaturization. The variance in detected transcripts was minimal and not influenced by the miniaturization level. Our results suggest that the workflow is highly reproducible and the sequence data suitable for downstream analyses such as differential gene expression analysis or variant calling. [ABSTRACT FROM AUTHOR]

Abstract Background Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method’s flexibility to a broader range of viruses, both DNA and RNA. Results Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. Conclusion TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses.

BackgroundNext generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.ResultsIn this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data.ConclusionsWe successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high‐throughput sequencing is becoming more cost‐efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA sources. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low‐coverage WGS from 60 DNA libraries prepared from four American Robin (Turdus migratorius) and four Abyssinian Thrush (Turdus abyssinicus) specimens collected in the 1920s. We compared DNA source (toepad versus incision‐line skin clip) and three library preparation methods: (1) double‐stranded DNA (dsDNA), single tube (KAPA); (2) single‐stranded DNA (ssDNA), multi‐tube (IDT); and (3) ssDNA, single tube (Claret Bioscience). We found that the ssDNA, multi‐tube method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best‐performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa. [ABSTRACT FROM AUTHOR]

Objective: Metatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (< 5 ng) with a library preparation kit (NEBNext Ultra II Directional RNA Library Prep Kit). Results: Good-quality RNA libraries were obtained from 0.1 ng of mock community and table surface RNA by changing the adapter concentration and number of PCR cycles. Differences in the target species of the rRNA depletion method affected the community composition and sensitivity of virus detection. The percentage of viral occupancy in two replicates was 0.259 and 0.290% in both human and bacterial rRNA-depleted samples, a 3.4 and 3.8-fold increase compared with that for only bacterial rRNA-depleted samples. Comparison of SARS-CoV-2 spiked-in human rRNA and bacterial rRNA-depleted samples suggested that more SARS-CoV-2 reads were detected in bacterial rRNA-depleted samples. We demonstrated that metatranscriptome analysis of RNA viromes is possible from RNA isolated from an indoor surface (representing a built-environment surface) using a standard library preparation kit. [ABSTRACT FROM AUTHOR]

Samples from avian species of high conservation concern are often low in total genomic DNA (gDNA). Feathers are collected more frequently than blood, yet they contain low total gDNA which can present challenges in using them for genomics. Whole Genome Sequencing (WGS) can provide many insights that can be used to aid in species conservation, but current methods for working with feathers are cost prohibitive for population level genomic analyses. Thus, there is a need for a cost-effective method of preparing WGS libraries from feathers. To bridge the gap between sampling techniques commonly used in avian conservation genetics that yield low total gDNA and the powerful tool of WGS, we developed a method that successfully produces WGS libraries from feather gDNA with as low input as 0.48 ng of DNA and an average final library size of 300–500 base pairs. Sequencing results suggest high sequencing quality using our protocol with feather DNA. We conclude that our method will facilitate high-throughput WGS on low total gDNA samples, like feathers, thus expanding the power of genomic tools in critical avian conservation research. [ABSTRACT FROM AUTHOR]

High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3′ termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) – a novel RNA-seq library preparation method directed towards mRNA 3′ termini. We demonstrate the specific enrichment for 3′-terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates.

Abstract Background Shotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to efficiently convert sub-nanogram DNA to high-quality metagenomic libraries and obtain high-fidelity data, hindering the exploration of niches with low microbial biomass. Results To cope with this challenge comprehensively, we evaluated the performance of various library preparation methods on 0.5 pg–5 ng synthetic microbial community DNA, characterized contaminants, and further applied different in silico decontamination methods. First, we discovered that whole genome amplification prior to library construction led to worse outcomes than preparing libraries directly. Among different non-WGA-based library preparation methods, we found the endonuclease-based method being generally good for different amounts of template and the tagmentation-based method showing specific advantages with 0.5 pg template, based on evaluation metrics including fidelity, proportion of designated reads, and reproducibility. The load of contaminating DNA introduced by library preparation varied from 0.01 to 15.59 pg for different kits and accounted for 0.05 to 45.97% of total reads. A considerable fraction of the contaminating reads were mapped to human commensal and pathogenic microbes, thus potentially leading to erroneous conclusions in human microbiome studies. Furthermore, the best performing in silico decontamination method in our evaluation, Decontam-either, was capable of recovering the real microbial community from libraries where contaminants accounted for less than 10% of total reads, but not from libraries with heavy and highly varied contaminants. Conclusions This study demonstrates that high-quality metagenomic data can be obtained from samples with sub-nanogram microbial DNA by combining appropriate library preparation and in silico decontamination methods and provides a general reference for method selection for samples with varying microbial biomass.

Abstract Background Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. Results In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. Conclusions We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.

This project evaluated the prototype PowerSeq® 46GY System using donor DNA and casework-type samples. The goal of this study was to determine whether modifications to the manufacturer's protocol could increase read coverage and improve sample results. Buccal and casework-type libraries were prepared using the TruSeq® DNA PCR-Free HT kit or the KAPA HyperPrep kit. Both kits were evaluated unmodified, and by substituting AMPure® XP beads for the beads of the most optimal kit. Two qPCR kits, the PowerSeq® Quant MS System and KAPA Library Quantification Kit, were also evaluated along with a KAPA size-adjustment workbook, which was compared as a third quantification method. Libraries were sequenced using the MiSeq® FGx and data were analyzed with STRait Razor. Results suggested that all three quantification methods overestimated library concentration, but the PowerSeq kit was most accurate. Samples prepared with the TruSeq library kit provided the highest coverage and the fewest instances of dropout and below-threshold alleles compared with the KAPA kit. Additionally, all bone and hair samples demonstrated full profile completeness, with bone samples yielding a higher average coverage than hair samples. Overall, our study demonstrated that the 46GY manufacturer's protocol produced the best quality results compared to alternative library preparation options. [ABSTRACT FROM AUTHOR]

Background: Previously developed TaME-seq method for deep sequencing of HPV, allowed simultaneous identification of the human papillomavirus (HPV) DNA consensus sequence, low-frequency variable sites, and chromosomal integration events. The method has been successfully validated and applied to the study of five carcinogenic high-risk (HR) HPV types (HPV16, 18, 31, 33, and 45). Here, we present TaME-seq2 with an updated laboratory workflow and bioinformatics pipeline. The HR-HPV type repertoire was expanded with HPV51, 52, and 59. As a proof-of-concept, TaME-seq2 was applied on SARS-CoV-2 positive samples showing the method's flexibility to a broader range of viruses, both DNA and RNA. Results: Compared to TaME-seq version 1, the bioinformatics pipeline of TaME-seq2 is approximately 40× faster. In total, 23 HPV-positive samples and seven SARS-CoV-2 clinical samples passed the threshold of 300× mean depth and were submitted to further analysis. The mean number of variable sites per 1 kb was ~ 1.5× higher in SARS-CoV-2 than in HPV-positive samples. Reproducibility and repeatability of the method were tested on a subset of samples. A viral integration breakpoint followed by a partial genomic deletion was found in within-run replicates of HPV59-positive sample. Identified viral consensus sequence in two separate runs was > 99.9% identical between replicates, differing by a couple of nucleotides identified in only one of the replicates. Conversely, the number of identical minor nucleotide variants (MNVs) differed greatly between replicates, probably caused by PCR-introduced bias. The total number of detected MNVs, calculated gene variability and mutational signature analysis, were unaffected by the sequencing run. Conclusion: TaME-seq2 proved well suited for consensus sequence identification, and the detection of low-frequency viral genome variation and viral-chromosomal integrations. The repertoire of TaME-seq2 now encompasses seven HR-HPV types. Our goal is to further include all HR-HPV types in the TaME-seq2 repertoire. Moreover, with a minor modification of previously developed primers, the same method was successfully applied for the analysis of SARS-CoV-2 positive samples, implying the ease of adapting TaME-seq2 to other viruses. [ABSTRACT FROM AUTHOR]

Abstract Museum specimens collected prior to cryogenic tissue storage are increasingly being used as genetic resources, and though high‐throughput sequencing is becoming more cost‐efficient, whole genome sequencing (WGS) of historical DNA (hDNA) remains inefficient and costly due to its short fragment sizes and high loads of exogenous DNA, among other factors. It is also unclear how sequencing efficiency is influenced by DNA sources. We aimed to identify the most efficient method and DNA source for collecting WGS data from avian museum specimens. We analyzed low‐coverage WGS from 60 DNA libraries prepared from four American Robin (Turdus migratorius) and four Abyssinian Thrush (Turdus abyssinicus) specimens collected in the 1920s. We compared DNA source (toepad versus incision‐line skin clip) and three library preparation methods: (1) double‐stranded DNA (dsDNA), single tube (KAPA); (2) single‐stranded DNA (ssDNA), multi‐tube (IDT); and (3) ssDNA, single tube (Claret Bioscience). We found that the ssDNA, multi‐tube method resulted in significantly greater endogenous DNA content, average read length, and sequencing efficiency than the other tested methods. We also tested whether a predigestion step reduced exogenous DNA in libraries from one specimen per species and found promising results that warrant further study. The ~10% increase in average sequencing efficiency of the best‐performing method over a commonly implemented dsDNA library preparation method has the potential to significantly increase WGS coverage of hDNA from bird specimens. Future work should evaluate the threshold for specimen age at which these results hold and how the combination of library preparation method and DNA source influence WGS in other taxa.

Abstract Small RNA (sRNA) profiling of Extracellular Vesicles (EVs) by Next‐Generation Sequencing (NGS) often delivers poor outcomes, independently of reagents, platforms or pipelines used, which contributes to poor reproducibility of studies. Here we analysed pre/post‐sequencing quality controls (QC) to predict issues potentially biasing biological sRNA‐sequencing results from purified human milk EVs, human and mouse EV‐enriched plasma and human paraffin‐embedded tissues. Although different RNA isolation protocols and NGS platforms were used in these experiments, all datasets had samples characterized by a marked removal of reads after pre‐processing. The extent of read loss between individual samples within a dataset did not correlate with isolated RNA quantity or sequenced base quality. Rather, cDNA electropherograms revealed the presence of a constant peak whose intensity correlated with the degree of read loss and, remarkably, with the percentage of adapter dimers, which were found to be overrepresented sequences in high read‐loss samples. The analysis through a QC pipeline, which allowed us to monitor quality parameters in a step‐by‐step manner, provided compelling evidence that adapter dimer contamination was the main factor causing batch effects. We concluded this study by summarising peer‐reviewed published workflows that perform consistently well in avoiding adapter dimer contamination towards a greater likelihood of sequencing success.

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Background: MicroRNAs (miRNAs) play an essential role in various biological processes and signaling pathways through the regulation of gene expression and genome stability. Recent data indicated that the next-generation sequencing (NGS)-based high-throughput quantification of miRNAs from biofluids provided exciting possibilities for discovering biomarkers of various diseases and might help promote the development of the early diagnosis of cancer. However, the complex process of library construction for sequencing always introduces bias, which may twist the actual expression levels of miRNAs and reach misleading conclusions. Results: We discussed the deviation issue in each step during constructing miRNA sequencing libraries and suggested many strategies to generate high-quality data by avoiding or minimizing bias. For example, improvement of adapter design (a blocking element away from the ligation end, a randomized fragment adjacent to the ligation junction and UMI) and optimization of ligation conditions (a high concentration of PEG 8000, reasonable incubation temperature and time, and the selection of ligase) in adapter ligation, high-quality input RNA samples, removal of adapter dimer (solid phase reverse immobilization (SPRI) magnetic bead, locked nucleic acid (LNA) oligonucleotide, and Phi29 DNA polymerase), PCR (linear amplification, touch-down PCR), and product purification are essential factors for achieving high-quality sequencing data. Moreover, we described several protocols that exhibit significant advantages using combinatorial optimization and commercially available low-input miRNA library preparation kits. Conclusions: Overall, our work provides the basis for unbiased high-throughput quantification of miRNAs. These data will help achieve optimal design involving miRNA profiling and provide reliable guidance for clinical diagnosis and treatment by significantly increasing the credibility of potential biomarkers. [ABSTRACT FROM AUTHOR]

The human genome is divided into functional units that replicate at specific times during S-phase. This temporal program is known as replication timing (RT) and is coordinated with the spatial organization of the genome and transcriptional activity. RT is also cell type–specific, dynamically regulated during development, and alterations in RT are observed in multiple diseases. Thus, the precise measure of RT is critical to understand the role of RT in gene function regulation. Distinct methods for assaying the RT program exist; however, conventional methods require thousands of cells as input, prohibiting its applicability to samples with limited cell numbers such as those from disease patients or from early developing embryos. Although single-cell RT analyses have been developed, these methods are low throughput, require generation of numerous libraries, increased sequencing costs, and produce low resolution data. Here, we developed an improved method to measure RT genome-wide that enables high-resolution analysis of low input samples. This method incorporates direct cell sorting into lysis buffer, as well as DNA fragmentation and library preparation in a single tube, resulting in higher yields, increased quality, and reproducibility with decreased costs. We also performed a systematic data processing analysis to provide standardized parameters for RT measurement. This optimized method facilitates RT analysis and will enable its application to a broad range of studies investigating the role of RT in gene expression, nuclear architecture, and disease. [ABSTRACT FROM AUTHOR]

RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits are proposed for FFPE samples yet no comparison on low quantities were performed. To select the most reliable, cost-effective, and relevant RNA-Seq approach, we applied five FFPE-compatible kits (based on 3′ capture, exome-capture and ribodepletion approaches) using 8 ng to 400 ng of FFPE-derived RNA and compared them to Nanostring on FFPE samples and to a reference PolyA (Truseq) approach on flash-frozen samples of the same tumors. We compared gene expression correlations and reproducibility. The Smarter Pico V3 ribodepletion approach appeared systematically the most comparable to Nanostring and Truseq (p < 0.001) and was a highly reproducible technique. In comparison with exome-capture and 3′ kits, the Smarter appeared more comparable to Truseq (p < 0.001). Overall, our results suggest that the Smarter is the most robust RNA-Seq technique to study small FFPE samples and 3′ Lexogen presents an interesting quality–price ratio for samples with less limiting quantities.

Abstract Background A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. Results Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ′ end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. Conclusions Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes.

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

Next-generation sequencing technologies have enabled many advances across biology, with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low-cost miniaturized (5-µl volume) high-throughput (384-sample) amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5-µl reaction volume with 0.2 µl of genomic DNA (gDNA) and the standard 25-µl reaction volume with 1 µl of gDNA. Between the two methods, alpha diversity was highly correlated (R 2 > 0.95), while distances of technical replicates were much lower than within-body-site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25-µl reactions to triplicate 5-µl reactions) are large enough to cover a MiSeq sequencing run for 768 samples while preserving accurate microbiome measurements. IMPORTANCE Reduced costs of sequencing have tremendously impacted the field of microbial ecology, allowing scientists to design more studies with larger sample sizes that often exceed 10,000 samples. Library preparation costs have not kept pace with sequencing prices, although automated liquid handling robots provide a unique opportunity to bridge this gap while also decreasing human error. Here, we take advantage of an acoustic liquid handling robot to develop a high-throughput miniaturized library preparation method of a highly cited and broadly used 16S rRNA gene amplicon reaction. We evaluate the potential negative effects of reducing the PCR volume along with varying the amount of gDNA going into the reaction. Our optimized method reduces sample-processing costs while continuing to generate a high-quality microbiome readout that is indistinguishable from the original method.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Background : Shotgun metagenomic sequencing has greatly expanded the understanding of microbial communities in various biological niches. However, it is still challenging to efficiently convert sub-nanogram DNA to high-quality metagenomic libraries and obtain high-fidelity data, hindering the exploration of niches with low microbial biomass. Results: To cope with this challenge comprehensively, we evaluated the performance of various library preparation methods on 0.5 pg–5 ng synthetic microbial community DNA, characterized contaminants, and further applied different in silico decontamination methods. First, we discovered that whole genome amplification prior to library construction led to worse outcomes than preparing libraries directly. Among different non-WGA-based library preparation methods, we found the endonuclease-based method being generally good for different amounts of template and the tagmentation-based method showing specific advantages with 0.5 pg template, based on evaluation metrics including fidelity, proportion of designated reads, and reproducibility. The load of contaminating DNA introduced by library preparation varied from 0.01 to 15.59 pg for different kits and accounted for 0.05 to 45.97% of total reads. A considerable fraction of the contaminating reads were mapped to human commensal and pathogenic microbes, thus potentially leading to erroneous conclusions in human microbiome studies. Furthermore, the best performing in silico decontamination method in our evaluation, Decontam-either, was capable of recovering the real microbial community from libraries where contaminants accounted for less than 10% of total reads, but not from libraries with heavy and highly varied contaminants. Conclusions: This study demonstrates that high-quality metagenomic data can be obtained from samples with sub-nanogram microbial DNA by combining appropriate library preparation and in silico decontamination methods and provides a general reference for method selection for samples with varying microbial biomass. [ABSTRACT FROM AUTHOR]

Background: Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. Results: In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. Conclusions: We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations. [ABSTRACT FROM AUTHOR]

Abstract Background Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations. Results We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. 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 the peaks by these short fragments. Conclusions We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

The COVID-19 pandemic demonstrated how rapidly various molecular methods can be adapted for a Public Health Emergency. Whether a need arises for whole-genome studies (next-generation sequencing), fast and high-throughput diagnostics (reverse-transcription real-time PCR) or global immunization (construction of mRNA or viral vector vaccines), the scientific community has been able to answer all these calls. In this study, we aimed at the assessment of effectiveness of the commercially available solution for full-genome SARS-CoV-2 sequencing (AmpliSeq™ SARS-CoV-2 Research Panel and Ion AmpliSeq™ Library Kit Plus, Thermo Fisher Scientific). The study is based on 634 samples obtained from patients from Poland, with varying viral load, assigned to a number of lineages. Here, we also present the results of protocol modifications implemented to obtain high-quality genomic data. We found that a modified library preparation protocol required less viral RNA input in order to obtain the optimal library quantity. Concurrently, neither concentration of cDNA nor reamplification of libraries from low-template samples improved the results of sequencing. On the basis of the amplicon success rates, we propose one amplicon to be redesigned, namely, the r1_1.15.1421280, for which less than 50 reads were produced by 44% of samples. Additionally, we found several mutations within different SARS-CoV-2 lineages that cause the neighboring amplicons to underperform. Therefore, due to constant SARS-CoV-2 evolution, we support the idea of conducting ongoing sequence-based surveillance studies to continuously validate commercially available RT-PCR and whole-genome sequencing solutions. [ABSTRACT FROM AUTHOR]

Spatial information of tissues is an essential component to reach a holistic overview of gene expression mechanisms. The sequencing-based Spatial transcriptomics approach allows to spatially barcode the whole transcriptome of tissue sections using microarray glass slides. However, manual preparation of high-quality tissue sequencing libraries is time-consuming and subjected to technical variability. Here, we present an automated adaptation of the 10x Genomics Visium library construction on the widely used Agilent Bravo Liquid Handling Platform. Compared to the manual Visium library preparation, our automated approach reduces hands-on time by over 80% and provides higher throughput and robustness. Our automated Visium library preparation protocol provides a new strategy to standardize spatially resolved transcriptomics analysis of tissues at scale.

Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample‐specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during "library preparation", that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample‐specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one‐step PCR, two‐step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications. [ABSTRACT FROM AUTHOR]

Background: A variety of protocols exist for producing whole genome run-on transcription datasets. However, little is known about how differences between these protocols affect the signal within the resulting libraries. Results: Using run-on transcription datasets generated from the same biological system, we show that a variety of GRO- and PRO-seq preparation methods leave identifiable signatures within each library. Specifically we show that the library preparation method results in differences in quality control metrics, as well as differences in the signal distribution at the 5 ′ end of transcribed regions. These shifts lead to disparities in eRNA identification, but do not impact analyses aimed at inferring the key regulators involved in changes to transcription. Conclusions: Run-on sequencing protocol variations result in technical signatures that can be used to identify both the enrichment and library preparation method of a particular data set. These technical signatures are batch effects that limit detailed comparisons of pausing ratios and eRNAs identified across protocols. However, these batch effects have only limited impact on our ability to infer which regulators underlie the observed transcriptional changes. [ABSTRACT FROM AUTHOR]

ABSTRACT Metagenomics is increasingly used to describe microbial communities in biological specimens. Ideally, the steps involved in the processing of the biological specimens should not change the microbiome composition in a way that it could lead to false interpretations of inferred microbial community composition. Common steps in sample preparation include sample collection, storage, DNA isolation, library preparation, and DNA sequencing. Here, we assess the effect of three library preparation kits and two DNA sequencing platforms. Of the library preparation kits, one involved a PCR step (Nextera), and two were PCR free (NEXTflex and KAPA). We sequenced the libraries on Illumina HiSeq and NextSeq platforms. As example microbiomes, two pig fecal samples and two sewage samples of which aliquots were stored at different storage conditions (immediate processing and storage at −80°C) were assessed. All DNA isolations were performed in duplicate, totaling 80 samples, excluding controls. We found that both library preparation and sequencing platform had systematic effects on the inferred microbial community composition. The different sequencing platforms introduced more variation than library preparation and freezing the samples. The results highlight that all sample processing steps need to be considered when comparing studies. Standardization of sample processing is key to generating comparable data within a study, and comparisons of differently generated data, such as in a meta-analysis, should be performed cautiously. IMPORTANCE Previous research has reported effects of sample storage conditions and DNA isolation procedures on metagenomics-based microbiome composition; however, the effect of library preparation and DNA sequencing in metagenomics has not been thoroughly assessed. Here, we provide evidence that library preparation and sequencing platform introduce systematic biases in the metagenomic-based characterization of microbial communities. These findings suggest that library preparation and sequencing are important parameters to keep consistent when aiming to detect small changes in microbiome community structure. Overall, we recommend that all samples in a microbiome study are processed in the same way to limit unwanted variations that could lead to false conclusions. Furthermore, if we are to obtain a more holistic insight from microbiome data generated around the world, we will need to provide more detailed sample metadata, including information about the different sample processing procedures, together with the DNA sequencing data at the public repositories.

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

High-throughput RNA sequencing offers a comprehensive analysis of transcriptome complexity originated from regulatory events, such as differential gene expression, alternative polyadenylation and others, and allows the increase in diagnostic capacity and precision. For gene expression profiling applications that do not specifically require information on alternative splicing events, the mRNA 3' termini counting approach is a cost-effective alternative to whole transcriptome sequencing. Here, we report MTAS-seq (mRNA sequencing via terminator-assisted synthesis) - a novel RNA-seq library preparation method directed towards mRNA 3' termini. We demonstrate the specific enrichment for 3'- terminal regions by simple and quick single-tube protocol with built-in molecular barcoding to enable accurate estimation of transcript abundance. To achieve that, we synthesized oligonucleotide-modified dideoxynucleotides which enable the generation of cDNA libraries at the reverse transcription step. We validated the performance of MTAS-seq on well-characterized reference bulk RNA and further tested it with eukaryotic cell lysates. [ABSTRACT FROM AUTHOR]

In the era of big data in Chemistry, the need for automated tools for virtual screening is compelling. Here, we present PrepFlow a toolkit for chemical library preparation and management. Starting from a list of compounds in SMILES or 2D molecular format, PrepFlow outputs a set of 3D molecular structures ready for use in subsequent drug discovery projects. Our development stands out for speed and robustness of execution, the efficient exploitation of HPC resources, and the implementation of an archiving strategy to save computer time, storage, and human intervention. Using a random selection of 600 compounds from available drug banks, we show that the preparation time per ligand on a desktop computer is 6.6 s. Thanks to these performances and the automatic parallelization on HPC, a chemical library of the size of ChEMBL (2 M) was prepared in around 3 days on a computer cluster. PrepFlow is freely distributed at the following link: https://ifm.chimie.unistra.fr/prepflow. [ABSTRACT FROM AUTHOR]

Abstract Background Next generation sequencing (NGS) has been widely used in biological research, due to its rapid decrease in cost and increasing ability to generate data. However, while the sequence generation step has seen many improvements over time, the library preparation step has not, resulting in low-efficiency library preparation methods, especially for the most time-consuming and labor-intensive steps: size-selection and quantification. Consequently, there can be bottlenecks in projects with large sample cohorts. Results We have described the all-in-one sequencing (AIO-seq) method, where instead of performing size-selection and quantification for samples individually, one sample one tube, up to 116 samples are pooled and analyzed in a single tube, ‘All-In-One’. The AIO-seq method pools libraries based on the samples’ expected data yields and the calculated concentrations of the size selected regions (target region), which can easily be obtained with the Agilent 2100 Bioanalyzer and Qubit Fluorometer. AIO-seq was applied to whole genome sequencing and RNA-seq libraries successfully, and it is envisaged that it could be applied to any type of NGS library, such as chromatin immunoprecipitation coupled with massively parallel sequencing, assays for transposase-accessible chromatin with high-throughput sequencing, and high-throughput chromosome conformation capture. We also demonstrated that for genetic population samples with low coverage sequences, like recombinant inbred lines (RIL), AIO-seq could be further simplified, by mixing the libraries immediately after PCR, without calculating the target region concentrations. Conclusions The AIO-seq method is thus labor saving and cost effective, and suitable for projects with large sample cohorts, like those used in plant breeding or population genetics research.

Abstract Background Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. Results In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. Conclusions We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.