Multi-omics approach dissects cis-regulatory mechanisms underlying North Carolina macular dystrophy, a retinal enhanceropathy.

North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

IS-Seq: a bioinformatics pipeline for integration sites analysis with comprehensive abundance quantification methods.

BACKGROUND: Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline. RESULTS: Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems. CONCLUSIONS: We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation.

UMI-77 Modulates the Complement Cascade Pathway and Inhibits Inflammatory Factor Storm in Sepsis Based on TMT Proteomics and Inflammation Array Glass Chip.

Sepsis is a systemic inflammatory response syndrome caused by infection, which has no specific drug at present. UMI-77 can significantly improve the survival rate of septic mice; the detailed role of UMI-77 and its underlying mechanisms in sepsis are not clear. Inflammation array glass chip and proteomic analyses were performed to elucidate the latent mechanism of UMI-77 in the treatment of sepsis. The results showed that 7.0 mg/kg UMI-77 improved the 5 day survival rate in septic mice compared to the LPS group (60.964 vs 9.779%) and ameliorated the pathological conditions. Inflammation array glass chip analysis showed that sepsis treatment with UMI-77 may eventually through the suppression of the characteristic inflammatory storm-related cytokines such as KC, RANTES, LIX, IL-6, eotaxin, TARC, IL-1ß, and so on. Proteomics analysis showed that 213 differential expression proteins and complement and coagulation cascades were significantly associated with the process for the UMI-77 treatment of sepsis. The top 10 proteins including Apoa2, Tgfb1, Serpinc1, Vtn, Apoa4, Cat, Hp, Serpinf2, Fgb, and Serpine1 were identified and verified, which play important roles in the mechanism of UMI-77 in the treatment of sepsis. Our findings indicate that UMI-77 exerts an antisepsis effect by modulating the complement cascade pathway and inhibiting inflammatory storm factors.

Pilot study of a comprehensive resource estimation method from environmental DNA using universal D-loop amplification primers.

Many studies have investigated the ability of environmental DNA (eDNA) to identify the species. However, when individual species are to be identified, accurate estimation of their abundance using traditional eDNA analyses is still difficult. We previously developed a novel analytical method called HaCeD-Seq (haplotype count from eDNA by sequencing), which focuses on the mitochondrial D-loop sequence for eels and tuna. In this study, universal D-loop primers were designed to enable the comprehensive detection of multiple fish species by a single sequence. To sequence the full-length D-loop with high accuracy, we performed nanopore sequencing with unique molecular identifiers (UMI). In addition, to determine the D-loop reference sequence, whole genome sequencing was performed with thin coverage, and complete mitochondrial genomes were determined. We developed a UMI-based Nanopore D-loop sequencing analysis pipeline and released it as open-source software. We detected 5 out of 15 species (33%) and 10 haplotypes out of 35 individuals (29%) among the detected species. This study demonstrates the possibility of comprehensively obtaining information related to population size from eDNA. In the future, this method can be used to improve the accuracy of fish resource estimation, which is currently highly dependent on fishing catches.

Benefits of applying molecular barcoding systems are not uniform across different genomic applications.

BACKGROUND: Despite the wide variety of Next Generation Sequencing (NGS)-based methods, it remains challenging to detect mutations present at very low frequencies. This problem is particularly relevant in oncology, where the limiting amount of input material, and its low quality, often limit the performance of the assays. Unique Molecular Identifiers (UMIs) are a molecular barcoding system often coupled with computational methods of noise suppression to improve the reliability of detection of rare variants. Although widely adopted, UMI inclusion imposes additional technical complexity and sequencing cost. Currently, there are no guidelines on UMI usage nor a comprehensive evaluation of their advantage across different applications. METHODS: We used DNA sequencing data generated by molecular barcoding and hybridization-based enrichment, from various types and quantities of input material (fresh frozen, formaldehyde-treated and cell-free DNA), to evaluate the performance of variant calling in different clinically relevant contexts. RESULTS: Noise suppression achieved by read grouping based on fragment mapping positions ensures reliable variant calling for many experimental designs even without exogenous UMIs. Exogenous barcodes significantly improve performance only when mapping position collisions occur, which is common in cell-free DNA. CONCLUSIONS: We demonstrate that UMI usage is not universally beneficial across experimental designs and that it is worthwhile to critically consider the comparative advantage of UMI usage for a given NGS application prior to experimental design.

Polyhydroxybutyrate production by recombinant Escherichia coli based on genes related to synthesis pathway of PHB from Massilia sp. UMI-21.

BACKGROUND: Polyhydroxybutyrate (PHB) is currently the most common polymer produced by natural bacteria and alternative to conventional petrochemical-based plastics due to its similar material properties and biodegradability. Massilia sp. UMI-21, a newly found bacterium, could produce PHB from starch, maltotriose, or maltose, etc. and could serve as a candidate for seaweed-degrading bioplastic producers. However, the genes involved in PHB metabolism in Massilia sp. UMI-21 are still unclear. RESULTS: In the present study, we assembled and annotated the genome of Massilia sp. UMI-21, identified genes related to the metabolism of PHB, and successfully constructed recombinant Escherichia coli harboring PHB-related genes (phaA2, phaB1 and phaC1) of Massilia sp. UMI-21, which showed up to 139.41% more product. Also, the vgb gene (encoding Vitreoscilla hemoglobin) was introduced into the genetically engineered E. coli and gained up to 117.42% more cell dry weight, 213.30% more PHB-like production and 44.09% more product content. Fermentation products extracted from recombinant E. coli harboring pETDuet1-phaA2phaB1-phaC1 and pETDuet1-phaA2phaB1-phaC1-vgb were identified as PHB by Fourier Transform Infrared and Proton nuclear magnetic resonance spectroscopy analysis. Furthermore, the decomposition temperature at 10% weight loss of PHB extracted from Massilia sp. UMI-21, recombinant E. coli DH5α-pETDuet1-phaA2phaB1-phaC1 and DH5α-pETDuet1-phaA2phaB1-phaC1-vgb was 276.5, 278.7 and 286.3 °C, respectively, showing good thermal stability. CONCLUSIONS: Herein, we presented the whole genome information of PHB-producing Massilia sp. UMI-21 and constructed novel recombinant strains using key genes in PHB synthesis of strain UMI-21 and the vgb gene. This genetically engineered E. coli strain can serve as an effective novel candidate in E. coli cell factory for PHB production by the rapid cell growth and high PHB production.

Investigating UAV-Based Applications in Indoor-Outdoor Sports Stadiums and Open-Air Gatherings for Different Interference Conditions beyond 5G Networks.

With the onset of 5G technology, the number of users is increasing drastically. These increased numbers of users demand better service on the network. This study examines the millimeter wave bands working frequencies. Working in the millimeter wave band has the disadvantage of interference. This study aims to analyze the impact of different interference conditions on unmanned aerial vehicle use scenarios, such as open-air gatherings and indoor-outdoor sports stadiums. Performance analysis was carried out in terms of received power and path loss readings.

Improving high-resolution copy number variation analysis from next generation sequencing using unique molecular identifiers.

BACKGROUND: Recently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct. RESULTS: Here, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization. CONCLUSION: We provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

Application of the 3' mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue.

BACKGROUND: Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3' mRNA-seq method sequences the 3' region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3' mRNA-Seq using Lexogen QuantSeq 3' mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. RESULTS: The total mapped reads of QuantSeq 3' mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3' mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3' mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). CONCLUSION: In this study, QuantSeq 3' mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3' mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

WASP: a versatile, web-accessible single cell RNA-Seq processing platform.

BACKGROUND: The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software. RESULTS: Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources. CONCLUSIONS: With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP's post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.

Perspectives and Benefits of High-Throughput Long-Read Sequencing in Microbial Ecology.

Short-read, high-throughput sequencing (HTS) methods have yielded numerous important insights into microbial ecology and function. Yet, in many instances short-read HTS techniques are suboptimal, for example, by providing insufficient phylogenetic resolution or low integrity of assembled genomes. Single-molecule and synthetic long-read (SLR) HTS methods have successfully ameliorated these limitations. In addition, nanopore sequencing has generated a number of unique analysis opportunities, such as rapid molecular diagnostics and direct RNA sequencing, and both Pacific Biosciences (PacBio) and nanopore sequencing support detection of epigenetic modifications. Although initially suffering from relatively low sequence quality, recent advances have greatly improved the accuracy of long-read sequencing technologies. In spite of great technological progress in recent years, the long-read HTS methods (PacBio and nanopore sequencing) are still relatively costly, require large amounts of high-quality starting material, and commonly need specific solutions in various analysis steps. Despite these challenges, long-read sequencing technologies offer high-quality, cutting-edge alternatives for testing hypotheses about microbiome structure and functioning as well as assembly of eukaryote genomes from complex environmental DNA samples.

Synthesis and evaluation of a UMI-77-based fluorescent probe for selective detecting Mcl-1 protein and imaging in living cancer cells.

Development of efficient fluorescent probes for detecting the overexpressed Mcl-1 protein in living cells is imperative for the diagnosis and treatment of cancers. In this paper, a new UMI-77 based fluorescent probe (DNSH), was synthesized and characterized. DNSH bound to the hydrophobic pockets of Mcl-1 protein tightly and the binding affinity was 20-fold higher than that of previous developed Mcl-1 probe. DNSH exhibited specific fluorescence response to Mcl-1 protein rather than other proteins. In the presence of Mcl-1 protein, fluorescence emission of DNSH can be switched on. Furthermore, fluorescence colocalization experiment demonstrated that DNSH can be successfully used for imaging mitochondrial Mcl-1 protein in human prostate cancer cells without a washing process. These results showed that DNSH may find useful applications in biological research such as tracking Mcl-1 protein in living biological specimens.

Ultrasensitive DNA Immune Repertoire Sequencing Using Unique Molecular Identifiers.

BACKGROUND: Immune repertoire sequencing of the T-cell receptor can identify clonotypes that have expanded as a result of antigen recognition or hematological malignancies. However, current sequencing protocols display limitations with nonuniform amplification and polymerase-induced errors during sequencing. Here, we developed a sequencing method that overcame these issues and applied it to γδ T cells, a cell type that plays a unique role in immunity, autoimmunity, homeostasis of intestine, skin, adipose tissue, and cancer biology. METHODS: The ultrasensitive immune repertoire sequencing method used PCR-introduced unique molecular identifiers. We constructed a 32-panel assay that captured the full diversity of the recombined T-cell receptor delta loci in γδ T cells. The protocol was validated on synthetic reference molecules and blood samples of healthy individuals. RESULTS: The 32-panel assay displayed wide dynamic range, high reproducibility, and analytical sensitivity with single-nucleotide resolution. The method corrected for sequencing-depended quantification bias and polymerase-induced errors and could be applied to both enriched and nonenriched cells. Healthy donors displayed oligoclonal expansion of γδ T cells and similar frequencies of clonotypes were detected in both enrichment and nonenriched samples. CONCLUSIONS: Ultrasensitive immune repertoire sequencing strategy enables quantification of individual and specific clonotypes in a background that can be applied to clinical as well as basic application areas. Our approach is simple, flexible, and can easily be implemented in any molecular laboratory.

Systematic assessment of commercially available low-input miRNA library preparation kits.

High-throughput sequencing is increasingly favoured to assay the presence and abundance of microRNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. Although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers (UMI) sequence tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of synthetic miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the relative levels of the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

scruff: an R/Bioconductor package for preprocessing single-cell RNA-sequencing data.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) enables the high-throughput quantification of transcriptional profiles in single cells. In contrast to bulk RNA-seq, additional preprocessing steps such as cell barcode identification or unique molecular identifier (UMI) deconvolution are necessary for preprocessing of data from single cell protocols. R packages that can easily preprocess data and rapidly visualize quality metrics and read alignments for individual cells across multiple samples or runs are still lacking. RESULTS: Here we present scruff, an R/Bioconductor package that preprocesses data generated from the CEL-Seq or CEL-Seq2 protocols and reports comprehensive data quality metrics and visualizations. scruff rapidly demultiplexes, aligns, and counts the reads mapped to genome features with deduplication of unique molecular identifier (UMI) tags. scruff also provides novel and extensive functions to visualize both pre- and post-alignment data quality metrics for cells from multiple experiments. Detailed read alignments with corresponding UMI information can be visualized at specific genome coordinates to display differences in isoform usage. The package also supports the visualization of quality metrics for sequence alignment files for multiple experiments generated by Cell Ranger from 10X Genomics. scruff is available as a free and open-source R/Bioconductor package. CONCLUSIONS: scruff streamlines the preprocessing of scRNA-seq data in a few simple R commands. It performs data demultiplexing, alignment, counting, quality report and visualization systematically and comprehensively, ensuring reproducible and reliable analysis of scRNA-seq data.

UTAP: User-friendly Transcriptome Analysis Pipeline.

BACKGROUND: RNA-Seq technology is routinely used to characterize the transcriptome, and to detect gene expression differences among cell types, genotypes and conditions. Advances in short-read sequencing instruments such as Illumina Next-Seq have yielded easy-to-operate machines, with high throughput, at a lower price per base. However, processing this data requires bioinformatics expertise to tailor and execute specific solutions for each type of library preparation. RESULTS: In order to enable fast and user-friendly data analysis, we developed an intuitive and scalable transcriptome pipeline that executes the full process, starting from cDNA sequences derived by RNA-Seq [Nat Rev Genet 10:57-63, 2009] and bulk MARS-Seq [Science 343:776-779, 2014] and ending with sets of differentially expressed genes. Output files are placed in structured folders, and results summaries are provided in rich and comprehensive reports, containing dozens of plots, tables and links. CONCLUSION: Our User-friendly Transcriptome Analysis Pipeline (UTAP) is an open source, web-based intuitive platform available to the biomedical research community, enabling researchers to efficiently and accurately analyse transcriptome sequence data.

Performance evaluation of commercial library construction kits for PCR-based targeted sequencing using a unique molecular identifier.

BACKGROUND: Target enrichment is a critical component of targeted deep next-generation sequencing for the cost-effective and sensitive detection of mutations, which is predominantly performed by either hybrid selection or PCR. Despite the advantages of efficient enrichment, PCR-based methods preclude the identification of PCR duplicates and their subsequent removal. Recently, this limitation was overcome by assigning a unique molecular identifier(UMI) to each template molecule. Currently, several commercial library construction kits based on PCR enrichment are available for UMIs, but there have been no systematic studies to compare their performances. In this study, we evaluated and compared the performances of five commercial library kits from four vendors: the Archer® Reveal ctDNA™ 28 Kit, NEBNext Direct® Cancer HotSpot Panel, Nugen Ovation® Custom Target Enrichment System, Qiagen Human Comprehensive Cancer Panel(HCCP), and Qiagen Human Actionable Solid Tumor Panel(HASTP). RESULTS: We evaluated and compared the performances of the five kits using 50 ng of genomic DNA for the library construction in terms of the library complexity, coverage uniformity, and errors in the UMIs. While the duplicate rates for all kits were dramatically decreased by identifying unique molecules with UMIs, the Qiagen HASTP achieved the highest library complexity based on the depth of unique coverage indicating superb library construction efficiency. Regarding the coverage uniformity, the kits from Nugen and NEB performed the best followed by the kits from Qiagen. We also analyzed the UMIs, including errors, which allowed us to adjust the depth of unique coverage and the length required for sufficient complexity. Based on these comparisons, we selected the Qiagen HASTP for further performance evaluations. The targeted deep sequencing method based on PCR target enrichment combined with UMI tagging sensitively detected mutations present at a frequency as low as 1% using 6.25 ng of human genomic DNA as the starting material. CONCLUSION: This study is the first systematic evaluation of commercial library construction kits for PCR-based targeted deep sequencing utilizing UMIs. Because the kits displayed significant variability in different quality metrics, our study offers a practical guideline for researchers to choose appropriate options for PCR-based targeted sequencing and useful benchmark data for evaluating new kits.

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

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

Unique, dual-indexed sequencing adapters with UMIs effectively eliminate index cross-talk and significantly improve sensitivity of massively parallel sequencing.

BACKGROUND: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. RESULTS: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. CONCLUSIONS: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.