PCR Duplicates Research Articles

A Novel Next-Generation Sequencing Assay for the Identification of BCR::ABL1 Transcript Type and Accurate and Sensitive Detection of TKI-Resistant Mutations.

The clinical management of chronic myeloid leukemia (CML) patients requires the identification of the type of BCR::ABL1 transcript at diagnosis and the monitoring of its expression and potential tyrosine kinase inhibitor (TKI) resistance mutations during treatment. Detection of resistant mutation requires transcript type-specific amplification of BCR::ABL1 from RNA. In this study, a custom RNA-based next-generation sequencing (NGS) assay (Dup-Seq BCR::ABL1) that enables (a) the identification of BCR::ABL1 transcript type and (b) the detection of resistance mutations from common and atypical BCR::ABL1 transcript types was developed and validated. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification for error correction. The custom data analysis pipeline enables breakpoint determination and overlapped mutation calling from duplicates, which minimizes the low-level mutation artifacts. This study demonstrates that this novel assay achieves high accuracy (positive percent agreement (PPA) for fusion: 98.5%; PPA and negative percent agreement (NPA) for mutation at 97.8% and 100.0%, respectively) and sensitivity (limit of detection (LOD) for mutation detection at 3% from 10 000 copies of BCR::ABL1 input). The Dup-Seq BCR::ABL1 assay not only allows for the identification of BCR::ABL1 typical and atypical transcript types and accurate and sensitive detection of TKI-resistant mutations but also simplifies molecular testing work flow for the clinical management of CML patients.

Pairtools: From sequencing data to chromosome contacts.

The field of 3D genome organization produces large amounts of sequencing data from Hi-C and a rapidly-expanding set of other chromosome conformation protocols (3C+). Massive and heterogeneous 3C+ data require high-performance and flexible processing of sequenced reads into contact pairs. To meet these challenges, we present pairtools-a flexible suite of tools for contact extraction from sequencing data. Pairtools provides modular command-line interface (CLI) tools that can be flexibly chained into data processing pipelines. The core operations provided by pairtools are parsing of.sam alignments into Hi-C pairs, sorting and removal of PCR duplicates. In addition, pairtools provides auxiliary tools for building feature-rich 3C+ pipelines, including contact pair manipulation, filtration, and quality control. Benchmarking pairtools against popular 3C+ data pipelines shows advantages of pairtools for high-performance and flexible 3C+ analysis. Finally, pairtools provides protocol-specific tools for restriction-based protocols, haplotype-resolved contacts, and single-cell Hi-C. The combination of CLI tools and tight integration with Python data analysis libraries makes pairtools a versatile foundation for a broad range of 3C+ pipelines.

Dissecting Selective Signatures and Candidate Genes in Grandparent Lines Subject to High Selection Pressure for Broiler Production and in a Local Russian Chicken Breed of Ushanka.

Breeding improvements and quantitative trait genetics are essential to the advancement of broiler production. The impact of artificial selection on genomic architecture and the genetic markers sought remains a key area of research. Here, we used whole-genome resequencing data to analyze the genomic architecture, diversity, and selective sweeps in Cornish White (CRW) and Plymouth Rock White (PRW) transboundary breeds selected for meat production and, comparatively, in an aboriginal Russian breed of Ushanka (USH). Reads were aligned to the reference genome bGalGal1.mat.broiler.GRCg7b and filtered to remove PCR duplicates and low-quality reads using BWA-MEM2 and bcftools software; 12,563,892 SNPs were produced for subsequent analyses. Compared to CRW and PRW, USH had a lower diversity and a higher genetic distinctiveness. Selective sweep regions and corresponding candidate genes were examined based on ZFST, hapFLK, and ROH assessment procedures. Twenty-seven prioritized chicken genes and the functional projection from human homologs suggest their importance for selection signals in the studied breeds. These genes have a functional relationship with such trait categories as body weight, muscles, fat metabolism and deposition, reproduction, etc., mainly aligned with the QTLs in the sweep regions. This information is pivotal for further executing genomic selection to enhance phenotypic traits.

Abstract 341: Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1

Abstract The clinical management of CML and Ph+ ALL patients requires the identification of BCR::ABL1 transcript variants and drug resistance mutations. Currently these assessments need to be performed using separate assays resulting in higher sample requirements, longer turnaround times and higher costs. In this study a custom NGS assay (Dup-Seq BCR-ABL1) was developed and validated that enables co-detection and identification of transcript variants (common and atypical) and resistance mutations. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification of BCR-ABL1 from a single source of the 1st strand cDNA initiated by ABL1 specific priming. The custom data analysis pipeline enables overlapped mutation calling from duplicates as well as transcript determination. The assay minimizes the low-level artifacts that can be seen with other NGS-based BCR::ABL1 assays. This study demonstrates that the Dup-Seq BCR-ABL1 assay achieves high accuracy (PPA for fusion: 0.985; PPA and NPA for mutation at 0.978 and 0.9999, respectively) and sensitivity (LoD for mutation detection at 3% from 10,000 copies of BCR-ABL1 input). The use of the Dup-Seq BCR-ABL1 assay for both transcript variant identification and mutation detection provides comprehensive and accurate results for the clinical management of CML and Ph+ ALL patients while reducing sample requirements, testing costs and turnaround time. Citation Format: Jin Li, Zhenyu Yan, Lin Shi, Wei Li, Weihua Liu, Cynthia Spittle, Chad Galderisi. Co-detection of BCR::ABL1 common and atypical fusion and mutation using a novel NGS assay, Dup-Seq BCR-ABL1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 341.

An overlooked phenomenon: complex interactions of potential error sources on the quality of bacterial de novo genome assemblies

BackgroundParameters adversely affecting the contiguity and accuracy of the assemblies from Illumina next-generation sequencing (NGS) are well described. However, past studies generally focused on their additive effects, overlooking their potential interactions possibly exacerbating one another’s effects in a multiplicative manner. To investigate whether or not they act interactively on de novo genome assembly quality, we simulated sequencing data for 13 bacterial reference genomes, with varying levels of error rate, sequencing depth, PCR and optical duplicate ratios.ResultsWe assessed the quality of assemblies from the simulated sequencing data with a number of contiguity and accuracy metrics, which we used to quantify both additive and multiplicative effects of the four parameters. We found that the tested parameters are engaged in complex interactions, exerting multiplicative, rather than additive, effects on assembly quality. Also, the ratio of non-repeated regions and GC% of the original genomes can shape how the four parameters affect assembly quality.ConclusionsWe provide a framework for consideration in future studies using de novo genome assembly of bacterial genomes, e.g. in choosing the optimal sequencing depth, balancing between its positive effect on contiguity and negative effect on accuracy due to its interaction with error rate. Furthermore, the properties of the genomes to be sequenced also should be taken into account, as they might influence the effects of error sources themselves.

Somatic mutations in tumor-adjacent normal breast tissue in young women with high risk family history.

576 Background: It’s incompletely understood why some women without germline breast cancer predisposing mutations develop early onset breast cancer. Breast cancer in young women is often more aggressive, with high histologic grade, worse prognosis, and higher risk of recurrence. We hypothesize that these women have somatic mutations in the normal breast tissue that contribute to breast cancer tumorigenesis. Methods: Whole exome sequencing (WES) data was analyzed from normal tissue adjacent to breast tumor and compared to matched peripheral blood (n=28). All patients were <50yo, had high risk family history as per NCCN guidelines, and had negative Ambry Genetics panel testing. FFPE sections were reviewed by two pathologists to identify normal breast tissue furthest away from tumor. WES was performed on Illumina HS4000 using 100-base pair paired-end reads. Samples were mapped to the human reference genome (vGRCh38). PCR duplicates were marked (Picard), and indel regions were adjusted (GATK). High-quality germline variants (HaplotypeCaller), somatic mutations (Mutect), and indels (IndelGenotyper) were called. Low-quality variants were filtered out. The classification and number of mutations per gene per sample were extracted and analyzed. Results: Majority of patients were white (86%), median age 45, with invasive ductal carcinoma (86%), ER+/PR+/HER2- (100%). Median somatic variants per sample were 97 (range: 45-285), all single nucleotide variants, mostly missense mutations, C>T conversions were seen in 45% of mutations. This suggests cytidine deaminase (APOBEC) involvement. All samples had alterations in at least one of the top ten affected genes which include transcription factors in zinc finger family (listed below). Zinc finger proteins play a key role in tumorigenesis, cancer progression and metastasis in a variety of cancers. Upregulation of zinc finger proteins has been associated with worse prognosis in breast cancer. Conclusions: We describe novel somatic mutations in zinc finger proteins in normal breast tissue adjacent to tumor that may contribute to early onset breast cancer in patients with high risk family history. [Table: see text]

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

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

Abstract A23: Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes

Abstract Malignant hematopoietic stem cells (HSCs) initiate the myelodysplastic syndromes (MDS) and drive treatment failure through their resistance to all non-transplant therapies. As a result, allogeneic stem cell transplantation (allo-SCT) is the only curative therapy for MDS. However, relapse remains the leading cause of treatment failure after allo-SCT. Improved tools to predict relapse in this setting are necessary to allow for pre-emptive therapeutic interventions, which may be more effective when the burden of disease is still low. Given prior evidence that HSCs serve as the functional unit of resistance to non-transplant therapies, we asked whether relapse after transplant originates from MDS HSCs, and whether their persistence can predict relapse. To overcome the limiting number of HSCs available in clinical bone marrow (BM) samples, we adapted a workflow developed for assessment of cell-free DNA and utilized a hybridization capture-based targeted sequencing panel covering 260 myeloid malignancy-associated genes. Unique dual index primers (UDIs) were utilized to tag input DNA, improving PCR duplicate identification, which overcome issues with amplification bias typically faced by low-input sequencing. This ultimately allowed us to consistently capture evaluable read depths from as few as 10 sorted cells. We assembled an initial cohort of 21 patients who underwent allo-SCT for MDS or secondary acute myeloid leukemia. Inclusion criteria were follow-up time greater than 1.5 years (or relapse in this timeframe), BM sampling prior to Day+120 post-transplant and relapse, and biospecimens allowing for sorting of greater than 10 HSCs. We sequenced sorted CD34+CD38-CD45RA- HSCs and multipotent progenitors (MPPs), CD34+CD38- CD45RA+ lymphoid-primed multipotent progenitors (LMPPs), and CD34+CD38+ progenitors, as well as bulk BM. With a median follow-up of two years, 9 of the 21 patients relapsed, with an average time to relapse of 362 days. Detection of disease mutations in HSCs/MPPs in any BM at D+30-D+120 post-transplant was 100% specific and 88% sensitive for relapse, showing improvements in both metrics over bulk BM assessment (22% sensitive and 66% specific), despite far lower cell input and read depth. Average time from detection to relapse was 6.9 months. In patients destined to relapse, disease mutations are often present in HSCs at a variant allele frequency of greater than 45%, while simultaneously undetectable on high-depth sequencing of bulk cells (>400x coverage). While even higher depth error-corrected sequencing bulk BM could likely detect such mutations, it would require >20x the sequencing depth, and could not identify the involved cell populations. In conclusion, we show for the first time that relapse of MDS after allo-SCT is driven by failure to eradicate MDS HSCs. Detection of MDS HSCs early post-transplant is highly predictive for relapse and opens new doors for pre-emptive interventions to prevent relapse. These findings are being validated in an independent cohort of 46 patients and updated data will be presented at the meeting. Citation Format: Benjamin Kroger, Yi Huang, Prapti Patel, Aimaz Afrough, Gurbakhash Kaur, Larry Anderson, Farrukh Awan, Praveen Ramakrishnan, Kunal Verma, Russell Vittrup, Robert H Collins, Madhuri Vusirikala, Yazan F Madanat, David Sallman, Carmelo Gurnari, Jaroslaw Maciejewski, Roni Tamari, Stephen Chung. Less is more: Ultra-low input sequencing allows for evaluation of stem cells as a predictor of post-transplant relapse in the myelodysplastic syndromes [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A23.

On the causes, consequences, and avoidance of PCR duplicates: Towards a theory of library complexity.

Library preparation protocols for most sequencing technologies involve PCR amplification of the template DNA, which open the possibility that a given template DNA molecule is sequenced multiple times. Reads arising from this phenomenon, known as PCR duplicates, inflate the cost of sequencing and can jeopardize the reliability of affected experiments. Despite the pervasiveness of this artefact, our understanding of its causes and of its impact on downstream statistical analyses remains essentially empirical. Here, we develop a general quantitative model of amplification distortions in sequencing data sets, which we leverage to investigate the factors controlling the occurrence of PCR duplicates. We show that the PCR duplicate rate is determined primarily by the ratio between library complexity and sequencing depth, and that amplification noise (including in its dependence on the number of PCR cycles) only plays a secondary role for this artefact. We confirm our predictions using new and published RAD-seq libraries and provide a method to estimate library complexity and amplification noise in any data set containing PCR duplicates. We discuss how amplification-related artefacts impact downstream analyses, and in particular genotyping accuracy. The proposed framework unites the numerous observations made on PCR duplicates and will be useful to experimenters of all sequencing technologies where DNA availability is a concern.

Evaluating the Transition from Targeted to Exome Sequencing: A Guide for Clinical Laboratories.

The transition from targeted to exome or genome sequencing in clinical contexts requires quality standards, such as targeted sequencing, in order to be fully adopted. However, no clear recommendations or methodology have emerged for evaluating this technological evolution. We developed a structured method based on four run-specific sequencing metrics and seven sample-specific sequencing metrics for evaluating the performance of exome sequencing strategies to replace targeted strategies. The indicators include quality metrics and coverage performance on gene panels and OMIM morbid genes. We applied this general strategy to three different exome kits and compared them with a myopathy-targeted sequencing method. After having achieved 80 million reads, all-tested exome kits generated data suitable for clinical diagnosis. However, significant differences in the coverage and PCR duplicates were observed between the kits. These are two main criteria to consider for the initial implementation with high-quality assurance. This study aims to assist molecular diagnostic laboratories in adopting and evaluating exome sequencing kits in a diagnostic context compared to the strategy used previously. A similar strategy could be used to implement whole-genome sequencing for diagnostic purposes.

Abstract 764: Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples

Abstract RNA-Seq is a revolutionary technology for transcriptome analysis, which has been used to quantify transcript levels, confirm gene annotation, identify novel transcripts, splice variants, and fusion detection. Traditionally, the usefulness of RNA-Seq has been limited by pre-analytical (sample availability, low integrity, tumor heterogeneity, complex protocols) and analytical factors (small mRNA fraction, high read duplicate rate), which can lead to higher costs, longer turnaround times, and false negative results. We have developed and automated a streamlined workflow for construction of highly complex RNA Libraries from fresh frozen and FFPE samples that addresses these concerns. We use target enrichment by bait selection with FFPE-derived samples, where rRNA depletion is found to be inconsistent and poly(A) enrichment cannot be used due to degradation of the source material. A fully automated protocol using pre-aliquoted reagents, has been optimized on the Magnis NGS Prep System for a wide range of RNA inputs (10-200 ng), various sample types (Intact and FFPE total RNA) and equipped with 192 Unique Dual sample Indices (UDI) to minimize the effects of possible index hopping and contamination. Addition of in-line molecular barcodes incorporated at the ligation step helps differentiate fragmentation from PCR duplicates, which increases the total number of reads available for further analysis (gene expression and fusion detection). With minimal risk of contamination, this automated protocol delivers up to eight target-enriched NGS Illumina sequencing-ready cDNA libraries from total RNA input per run in about 12 hours without the need of any user intervention. Both catalog and custom probes can be utilized, and PCR cycles can easily be adjusted from the touchscreen to optimize the library yields. For Research Use Only. Not for use in Diagnostic procedures. Citation Format: Saharnaz Bigdeli, Bahram Arezi, Jan Godoski, Denise Rhodes, Ryan Aguilar, Karen William, Bernd Buehler, Ji Zhu, Ronda Allen. Automated walk away NGS sample preparation for constructing in-line molecular barcoded RNA libraries from Fresh Frozen and FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 764.

PD25-12 IDENTIFICATION OF INTRATUMOR PATHOGENS FROM SINGLE-CELL RNA SEQUENCING, CELLULAR DYSFUNCTION AND IMMUNE RESPONSE

You have accessJournal of UrologyCME1 Apr 2023PD25-12 IDENTIFICATION OF INTRATUMOR PATHOGENS FROM SINGLE-CELL RNA SEQUENCING, CELLULAR DYSFUNCTION AND IMMUNE RESPONSE Laura Bukavina, Mohit Sindhani, Henkel Valentine, Daniel Ranti, Kirtishri Mishra, Alexander Kutikov, Shilpa Gupta, John Sfakianos, Mahmoud Ghannoum, Mauricio Retuerto, Andres Correa, Robert Uzzo, and Philip Abbosh Laura BukavinaLaura Bukavina More articles by this author , Mohit SindhaniMohit Sindhani More articles by this author , Henkel ValentineHenkel Valentine More articles by this author , Daniel RantiDaniel Ranti More articles by this author , Kirtishri MishraKirtishri Mishra More articles by this author , Alexander KutikovAlexander Kutikov More articles by this author , Shilpa GuptaShilpa Gupta More articles by this author , John SfakianosJohn Sfakianos More articles by this author , Mahmoud GhannoumMahmoud Ghannoum More articles by this author , Mauricio RetuertoMauricio Retuerto More articles by this author , Andres CorreaAndres Correa More articles by this author , Robert UzzoRobert Uzzo More articles by this author , and Philip AbboshPhilip Abbosh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pathogenic microbes can induce cellular dysfunction and alters immune response to therapy. Taking advantage of single-cell RNA-sequencing (scRNA-seq), we aim to assess the transcriptomic features of the single cell level to identify intracellular pathogens in patients with high grade bladder cancer. METHODS: CellRanger and Alevin were utilized to extract cell barcodes and unique molecular identifiers. Low complexity reads <20 was considered negative, and PCR duplicates were removed. Seuret Findmarkers, scIntegrate, and scDensity Plot were utilized to normalize the data, cluster the cells, and calculate markers in each cluster of differentially expressed genes between the two conditions. We focused our analysis on Pseudomonas aeruginosa due to high urogenital virulence, and immunomodulation capabilities by pattern recognition receptors and inflammasome. RESULTS: Patients with history of high-grade BC demonstrated increased presence of Aeromonas (175,473 reads, 54%), Pseudomonas (75,002 reads, 26.73%), and Bacillus (15,410 reads, 5.49%) in their tissue samples, not visualized in healthy controls (counts <10). Among 8,324 cells (across 7 patients), classical CD14+ monocyte subtype was most likely to be exposed to Pseudomonas species (4150 /5069, 81.8%), followed by non-classical CD16+ monocytes (722/1040, 69.4%), and CD8+ Tcells (348/680, 51.1%). (Figure 1A-C). Relative gene expression level between exposed and non-exposed monocytes demonstrated total 25 upregulated genes, with increased expression GNBL21 (aka: RACK1, log10 4.1), MALAT1(log10 3.7), TCEB2(log10 2.9) (increasing proliferation, migration, and reduction of cell autophagy) compared to non-infected, and normal controls. Pseudomonas involvement of non-monocyte cells (CD8+, CD4+Th17, CD4+ naïve) was associated with disease progression (2/5 patients), as well as increased expression of IFI6/27(interferon alpha inducible protein 6 & 27), ITAC (interferon inducible T cell alpha-chemoattractant). CONCLUSIONS: Altogether, our analysis depicted the distribution of intracellular bacteria not previously recognized within tumor microenvironment utilizing scRNAseq, and revealed overexpression of RACK1 in Pseudomonas positive cells, associated with apoptosis resistance. Source of Funding: Bladder Cancer Advocacy Network Young Investigator Award © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e734 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Laura Bukavina More articles by this author Mohit Sindhani More articles by this author Henkel Valentine More articles by this author Daniel Ranti More articles by this author Kirtishri Mishra More articles by this author Alexander Kutikov More articles by this author Shilpa Gupta More articles by this author John Sfakianos More articles by this author Mahmoud Ghannoum More articles by this author Mauricio Retuerto More articles by this author Andres Correa More articles by this author Robert Uzzo More articles by this author Philip Abbosh More articles by this author Expand All Advertisement PDF downloadLoading ...

DdRAD-seq variant calling in peach and the effect of removing PCR duplicates

ddRAD-seq variant calling in peach and the effect of removing PCR duplicates

System analysis of the sequencing quality of human whole exome samples on BGI NGS platform

Human exome sequencing is a classical method used in most medical genetic applications. The leaders in the field are the manufacturers of enrichment kits based on hybridization of cRNA or cDNA biotinylated probes specific for a genomic region of interest. Recently, the platforms manufactured by the Chinese company MGI Tech have become widespread in Europe and Asia. The reliability and quality of the obtained data are already beyond any doubt. However, only a few kits compatible with these sequencers can be used for such specific tasks as exome sequencing. We developed our own solution for library pre-capture pooling and exome enrichment with Agilent probes. In this work, using a set of the standard benchmark samples from the Platinum Genome collection, we demonstrate that the qualitative and quantitative parameters of our protocol which we called “RSMU_exome” exceed those of the MGI Tech kit. Our protocol allows for identifying more SNV and indels, generates fewer PCR duplicates, enables pooling of more samples in a single enrichment procedure, and requires less raw data to obtain results comparable with the MGI Tech's protocol. The cost of our protocol is also lower than that of MGI Tech's solution.

Replacing the SpCas9 HNH domain by deaminases generates compact base editors with an alternative targeting scope

Base editors are RNA-guided deaminases that enable site-specific nucleotide transitions. The targeting scope of these Cas-deaminase fusion proteins critically depends on the availability of a protospacer adjacent motif (PAM) at the target locus and is limited to a window within the CRISPR-Cas R-loop, where single-stranded DNA (ssDNA) is accessible to the deaminase. Here, we reason that the Cas9-HNH nuclease domain sterically constrains ssDNA accessibility and demonstrate that omission of this domain expands the editing window. By exchanging the HNH nuclease domain with a monomeric or heterodimeric adenosine deaminase, we furthermore engineer adenine base editor variants (HNHx-ABEs) with PAM-proximally shifted editing windows. This work expands the targeting scope of base editors and provides base editor variants that are substantially smaller. It moreover informs of potential future directions in Cas9 protein engineering, where the HNH domain could be replaced by other enzymes that act on ssDNA.

Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer

Background: The first step of virtually all next generation sequencing analysis involves the splitting of the raw sequencing data into separate files using sample-specific barcodes, a process known as “demultiplexing”. However, we found that existing software for this purpose was either too inflexible or too computationally intensive for fast, streamlined processing of raw, single end fastq files containing combinatorial barcodes. Results: Here, we introduce a fast and uniquely flexible demultiplexer, named Ultraplex, which splits a raw FASTQ file containing barcodes either at a single end or at both 5’ and 3’ ends of reads, trims the sequencing adaptors and low-quality bases, and moves unique molecular identifiers (UMIs) into the read header, allowing subsequent removal of PCR duplicates. Ultraplex is able to perform such single or combinatorial demultiplexing on both single- and paired-end sequencing data, and can process an entire Illumina HiSeq lane, consisting of nearly 500 million reads, in less than 20 minutes. Conclusions: Ultraplex greatly reduces computational burden and pipeline complexity for the demultiplexing of complex sequencing libraries, such as those produced by various CLIP and ribosome profiling protocols, and is also very user friendly, enabling streamlined, robust data processing. Ultraplex is available on PyPi and Conda and via Github.

Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer.

Background: The first step of virtually all next generation sequencing analysis involves the splitting of the raw sequencing data into separate files using sample-specific barcodes, a process known as "demultiplexing". However, we found that existing software for this purpose was either too inflexible or too computationally intensive for fast, streamlined processing of raw, single end fastq files containing combinatorial barcodes. Results: Here, we introduce a fast and uniquely flexible demultiplexer, named Ultraplex, which splits a raw FASTQ file containing barcodes either at a single end or at both 5' and 3' ends of reads, trims the sequencing adaptors and low-quality bases, and moves unique molecular identifiers (UMIs) into the read header, allowing subsequent removal of PCR duplicates. Ultraplex is able to perform such single or combinatorial demultiplexing on both single- and paired-end sequencing data, and can process an entire Illumina HiSeq lane, consisting of nearly 500 million reads, in less than 20 minutes. Conclusions: Ultraplex greatly reduces computational burden and pipeline complexity for the demultiplexing of complex sequencing libraries, such as those produced by various CLIP and ribosome profiling protocols, and is also very user friendly, enabling streamlined, robust data processing. Ultraplex is available on PyPi and Conda and via Github.

UMI4Cats: an R package to analyze chromatin contact profiles obtained by UMI-4C.

MotivationUMI-4C, a technique that combines chromosome conformation capture (4C) and unique molecular identifiers (UMI), is widely used to profile and quantitatively compare targeted chromosomal contact profiles. The analysis of UMI-4C experiments presents several computational challenges, including the removal of the PCR duplication bias and the identification of differential chromatin contacts.ResultsWe have developed UMI4Cats (UMI-4C Analysis Turned Simple), an R package that facilitates processing, analyzing and visualizing of data obtained by UMI-4C experiments.Availability and implementationUMI4Cats is implemented as an R package supported on Linux, MacOS and MS Windows. UMI4Cats is available from Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/UMI4Cats.html) and GitHub (https://github.com/Pasquali-lab/UMI4Cats).Supplementary information Supplementary data are available at Bioinformatics online.

A patient with human coronavirus NL63 falsely diagnosed with COVID-19; Lesson learned for the importance of definitive diagnosis

The gold standard for the diagnosis of coronavirus disease 2019 (COVID-19) is a nucleic acid detection test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may occasionally reveal false-positive or false-negative results. Herein, we describe a case of a patient infected with human coronavirus NL63 (HCoV-NL63) who was falsely diagnosed with COVID-19 using the Ampdirect™ 2019-nCoV detection kit (Shimadzu Corporation, Japan) and SARS-CoV-2 Detection Kit (TOYOBO co., ltd.), and was admitted to a COVID-19 hospital ward. We suspected a cross-reaction between HCoV-NL63 and SARS-CoV-2; however, the reported genome sequences of HCoV-NL63 and N1/N2 primers for SARS-CoV-2 do not correspond. Thus, the PCR result was supposed to be a false positive possibly due to contamination or human error. Although the issue of a false-negative result has been the focus of much attention to prevent the spread of the disease, a false positive is fraught with problems as well. Physicians should recognize that unnecessary isolation violates human rights and a careful diagnosis is indispensable when the results of laboratory testing for COVID-19 are unclear. Generally, in cases such as a duplicate PCR test was partially positive, either N1 or N2 alone was positive, PCR testing for two or more target regions resulted in a positive only for single region, a high cycle threshold >35 was obtained, a false positive should be suspected. Especially, when these conditions coincide, we should recognize the high likelihood of a false positive.

A head-to-head comparison of ribodepletion and polyA selection approaches for Caenorhabditis elegans low input RNA-sequencing libraries.

A recent and powerful technique is to obtain transcriptomes from rare cell populations, such as single neurons in Caenorhabditis elegans, by enriching dissociated cells using fluorescent sorting. However, these cell samples often have low yields of RNA that present challenges in library preparation. This can lead to PCR duplicates, noisy gene expression for lowly expressed genes, and other issues that limit endpoint analysis. Furthermore, some common resources, such as sequence-specific kits for removing ribosomal RNA, are not optimized for nonmammalian samples. To advance library construction for such challenging samples, we compared two approaches for building RNAseq libraries from less than 10 nanograms of C. elegans RNA: SMARTSeq V4 (Takara), a widely used kit for selecting poly-adenylated transcripts; and SoLo Ovation (Tecan Genomics), a newly developed ribodepletion-based approach. For ribodepletion, we used a custom kit of 200 probes designed to match C. elegans rRNA gene sequences. We found that SoLo Ovation, in combination with our custom C. elegans probe set for rRNA depletion, detects an expanded set of noncoding RNAs, shows reduced noise in lowly expressed genes, and more accurately counts expression of long genes. The approach described here should be broadly useful for similar efforts to analyze transcriptomics when RNA is limiting.