PCR Errors Research Articles

Unveiling errors in soil microbial community sequencing: a case for reference soils and improved diagnostics for nanopore sequencing

The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories.

Unique molecular identifier-based amplicon sequencing of microhaplotypes for background noise mitigation

Microhaplotypes (MHs), comprising two or more single-nucleotide polymorphisms in a short fragment, are promising forensic markers owing to their remarkable polymorphic nature. Several studies have demonstrated the utility of MHs through massively parallel sequencing (MPS). Nevertheless, the background noise level associated with MHs in MPS, which imposes a practical detection limit for the system, remains uninvestigated. Currently, unique molecular identifier (UMI) systems are known to effectively mitigate background noise by tracking original DNA molecules and facilitating PCR and MPS error corrections. Hence, this study aimed to design a UMI-based amplicon sequencing system, designated MH-UMIseq, which can amplify 46 MHs simultaneously and generate MPS libraries in four steps: barcoding PCR, nuclease reaction, boosting PCR, and indexing PCR. The performance of the MH-UMIseq system was evaluated using the Illumina NextSeq 550 and MiniSeq systems with 31 sets for 5 ng, 1 ng, and 200 pg of input DNA. The fgbio toolkit was used in conjunction with STRait Razor 3.0 and Visual Microhap to analyze the UMI data on MHs. The corresponding average not suppressed noise proportion of MH-UMIseq were 0.1 %, 0.3 %, and 0.7 % for 5 ng, 1 ng, and 200 pg of DNA, respectively, which substantially suppressed the background noise for more than 1 ng of DNA. Interestingly, the proportion of not suppressed noise in MH-UMIseq notably decreased as the amount of input DNA increased. The number of UMI families was proportional to the copy number of the template DNA and closely correlated with the system resolution. Therefore, the resolution of MH-UMIseq system is expected to be higher than that of conventional MPS for the deconvolution of mixtures containing more than 1 ng of DNA.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

Abstract 7425: UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules

Abstract The T-cell receptor (TCR) population in humans is comprised of highly diversified heterodimers, regulating the recognition of antigen-major histocompatibility complex. Tremendous TCR sequence diversity is produced by somatic recombination of several TCR gene loci each consisting of multiple gene segments. Next-generation sequencing has enabled comprehensive profiling of the TCR repertoire from different physiological and disease conditions ushering in much interest in using TCR-seq to assess T-cell diversity. However, during NGS library construction and sequencing, errors and enzymatic inefficiencies can compromise the accuracy of the final data, particularly around calling of the VD and VDJ recombined regions and subsequent clonotype assignment. To increase the accuracy of NGS sequencing, Unique Molecular Identifiers (UMIs), consisting of short random nucleotide bases, can be used to mark original molecules in NGS library allowing for error and bias corrections. There are two well studied technical limitations to apply UMIs: 1.) UMI sequences tend to collide when input molecule number is large 2.) UMI sequences are not insulated from PCR and sequencing errors. To address these limitations, many computational approaches had been published. Among them, very few can be used to solve UMI colliding errors and over-simplified error models were implemented for UMI sequencing error handling. Here we report a novel strategy and UMI structure which uses more complex UMIs that is longer and of different length. This results in minimizing UMI collision while maximizing sequencing quality. Our UMI analysis pipeline, “UMI-nea” is able to handle not only substitution errors but also indel errors and UMIs with different lengths. We developed a novel computational framework to parallelly process sequence comparisons to mitigate the elevated computational burden. To account for the varied dispersion of PCR efficiency for different molecules and error bearing UMIs from libraries with different input and with different sequencing depth, we also developed a statistical framework leveraging negative binomial model and single-cell knee plot to set a dynamic threshold for original molecule estimate. We verified UMI-nea with several simulated data and demonstrated that UMI-nea can achieve >99% completeness and homogeneity to recover the original molecule count with various error rates and UMI lengths, outperforming existing tools and methods in comparison. We applied UMI-nea to profile TCR for 8 PBMC samples sequenced on different Illumina platforms with different sequencing depths. We observed >85% reproducibility of clonotype calls on all samples. To test the sensitivity and specificity of UMI-nea, we sequenced pure cell line samples and cell line spike-in samples with different ratios and discovered very high recall and precision rates. Citation Format: Jixin Deng, Jingxiao Zhang, Song Tian, Samuel J. Rulli, Hong Xu, John DiCarlo, Eric Lader. UMI-nea: A fast and robust UMI analysis approach to accurately identify and quantify TCR repertoire from targeted RNA sequencing with wide range of input molecules [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 7425.

Correcting PCR amplification errors in unique molecular identifiers to generate accurate numbers of sequencing molecules

Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.

Longitudinal somatic mutation analysis of individual human CD8 +T cells by a UMI-based single cell mutation detection (USCMD) method

Abstract Somatic mutation is a contributing factor for cancer and aging. DNA-based single-cell mutation detection methods have been developed, but are hindered by the limited number of cells examined as well as by errors from PCR amplification and sequencing methods. Here, we developed a UMI-based scRNAseq mutation detection (USCMD) method that allows for the examination of up to 10,000 cells per sample with reduced PCR and sequencing errors, and which also filters potential errors of RNA polymerase II. We applied USCMD to analyze the scRNAseq data from human CD8 +T cells collected from 8 donors with 2 visits over a period of 9 years (aged from 30 to 69 years at first visit). We found that some novel missense mutations were associated with changes (increase or decrease) in the number of cells carrying them over time, and were able to identify clonally related cells based on shared somatic mutations. Together, this USCMD method improves the accuracy of mutational analysis of scRNAseq data and allows parallel transcriptional assessment of mutated and unmutated cells to understand their potential functional consequences.

Abstract 1042: Analytical validation of a liquid biopsy test using cell-free circulating tumor DNA for mutational profiling

Abstract Minimally invasive next-generation sequencing-based (NGS) liquid biopsy (LB) tests using cell-free DNA (cfDNA) are increasingly becoming an important tool for the clinical management of cancer patients. The ability to accurately detect molecular alterations from a single plasma sample is valuable for biomarker discovery, diagnostics, and disease monitoring. However, challenges for this assay include low concentrations of circulating tumor DNA (ctDNA) within the cfDNA plasma fraction and PCR and sequencing errors, which can lead to decreased sensitivity in variant calling. Further, somatic mosaicism due to clonal hematopoiesis of indeterminate potential (CHIP) in plasma makes accurate interpretation of LB results a challenge. Incorrect classification of false positives can lead to inappropriate therapeutic management. Here, we present an LB assay composed of a proprietary error reduction algorithm designed to eliminate false positives due to PCR and NGS errors and a white blood cell sequencing (WBC-seq) component to detect and bioinformatically filter out CHIP-related mutations. Consisting of a custom panel of 216 FDA/NCCN pan-cancer genes, our cfDNA LB assay was analytically validated with commercially available reference standards and human cell lines. At a clinically relevant sequencing depth of 4000x, we observed a limit of detection (LOD) of 0.2% variant allele frequency for single nucleotide variants (SNVs) and insertions and deletions (indels) with overall accuracies of 98% and 99% and sensitivities of 91% and 83%, respectively. To assess the LB assay performance on clinical samples, orthogonal whole-exome sequencing (WES) with subsequent standard somatic variant calling was performed on paired peripheral blood lymphocytes (PBLs) and plasma samples from 12 patients. The WES assay results were concordant with those obtained using our LB assay for SNVs (CCC = 0.75) and indels (CCC = 0.90). Further, our assay detected an additional 53% and 50% SNVs and indels, respectively, compared to WES. Finally, 8 out of 11 clinically actionable mutations were detected by both WES and the LB assay, while 3 additional SNVs were detected in the LB assay. Taken together, the use of WBC-seq to filter CHIP-derived mutations combined with our error reduction algorithm resulted in a substantial reduction in false positive variant calls without a compromise in the sensitivity or classification accuracy of SNVs and indels. Concordance was observed between traditional WES analysis and the LB assay developed here. These findings highlight the potential for our LB cfDNA assay to be used to improve personalized therapeutic options and guide clinical decision making. Citation Format: Anastasiya Yudina, Alexey Efremov, Danielle Sookiasian, Ekaterina Nuzhdina, Svetlana Podsvirova, Madison Chasse, Tori Conroy, Noel English, Sergey Starikov, Olesia Klimchuk, Dmitry Tabakov, Anna Love, Kushal Suryamohan, Artur Baisangurov, Cagdas Tazearslan, Nathan Fowler, Alexander Bagaev. Analytical validation of a liquid biopsy test using cell-free circulating tumor DNA for mutational profiling [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 1042.

The accuracy of reverse genetics systems for SARS-CoV-2: Circular polymerase extension reaction versus bacterial artificial chromosome.

Reverse genetics systems to rescue viruses from modified DNA are useful tools to investigate the molecular mechanisms of viruses. The COVID-19 pandemic prompted the development of several reverse genetics systems for SARS-CoV-2. The circular polymerase extension reaction (CPER) method enables the rapid generation of recombinant SARS-CoV-2; however, such PCR-based approaches could introduce unwanted mutations due to PCR errors. To compare the accuracy of CPER and a classic reverse genetics method using bacterial artificial chromosome (BAC), SARS-CoV-2 Wuhan/Hu-1/2019 was generated five times using BAC and five times using CPER. These 10 independent virus stocks were then deep sequencing, and the number of substitutions for which the frequency was greater than 10% was counted. No nucleotide substitutions with a frequency of greater than 10% were observed in all five independent virus stocks generated by the BAC method. In contrast, three to five unwanted nucleotide substitutions with a frequency of more than 10% were detected in four of the five virus stocks generated by the CPER. Furthermore, four substitutions with frequencies greater than 20% were generated in three virus stocks by using the CPER. We found that the accuracy of the CPER method is lower than that of the BAC method. Our findings suggest care should be used when employing the CPER method.

PURC Provides Improved Sequence Inference for Polyploid Phylogenetics and Other Manifestations of the Multiple-Copy Problem.

Inferring the true biological sequences from amplicon mixtures remains a difficult bioinformatics problem. The traditional approach is to cluster sequencing reads by similarity thresholds and treat the consensus sequence of each cluster as an "operational taxonomic unit" (OTU). Recently, this approach has been improved by model-based methods that correct PCR and sequencing errors in order to infer "amplicon sequence variants" (ASVs). To date, ASV approaches have been used primarily in metagenomics, but they are also useful for determining homeologs in polyploid organisms. To facilitate the usage of ASV methods among polyploidy researchers, we incorporated ASV inference alongside OTU clustering in PURC v2.0, a major update to PURC (Pipeline for Untangling Reticulate Complexes). In addition, PURC v2.0 features faster demultiplexing than the original version and has been updated to be compatible with Python 3. In this chapter we present results indicating that using the ASV approach is more likely to infer the correct biological sequences in comparison to the earlier OTU-based PURC and describe how to prepare sequencing data, run PURC v2.0 under several different modes, and interpret the output.

Using unique molecular identifiers to improve allele calling in low-template mixtures

PCR artifacts are an ever-present challenge in sequencing applications. These artifacts can seriously limit the analysis and interpretation of low-template samples and mixtures, especially with respect to a minor contributor. In medicine, molecular barcoding techniques have been employed to decrease the impact of PCR error and to allow the examination of low-abundance somatic variation. In principle, it should be possible to apply the same techniques to the forensic analysis of mixtures. To that end, several short tandem repeat loci were selected for targeted sequencing, and a bioinformatic pipeline for analyzing the sequence data was developed. The pipeline notes the relevant unique molecular identifiers (UMIs) attached to each read and, using machine learning, filters the noise products out of the set of potential alleles. To evaluate this pipeline, DNA from pairs of individuals were mixed at different ratios (1−1, 1−9) and sequenced with different starting amounts of DNA (10, 1 and 0.1 ng). Naïvely using the information in the molecular barcodes led to increased performance, with the machine learning resulting in an additional benefit. In concrete terms, using the UMI data results in less noise for a given amount of drop out. For instance, if thresholds are selected that filter out a quarter of the true alleles, using read counts accepts 2381 noise alleles and using raw UMI counts accepts 1726 noise alleles, while the machine learning approach only accepts 307.

Unique Molecular Identifiers and Multiplexing Amplicons Maximize the Utility of Deep Sequencing To Critically Assess Population Diversity in RNA Viruses.

Next generation sequencing (NGS)/deep sequencing has become an important tool in the study of viruses. The use of unique molecular identifiers (UMI) can overcome the limitations of PCR errors and PCR-mediated recombination and reveal the true sampling depth of a viral population being sequenced in an NGS experiment. This approach of enhanced sequence data represents an ideal tool to study both high and low abundance drug resistance mutations and more generally to explore the genetic structure of viral populations. Central to the use of the UMI/Primer ID approach is the creation of a template consensus sequence (TCS) for each genome sequenced. Here we describe a series of experiments to validate several aspects of the Multiplexed Primer ID (MPID) sequencing approach using the MiSeq platform. We have evaluated how multiplexing of cDNA synthesis and amplicons affects the sampling depth of the viral population for each individual cDNA and amplicon to understand the relationship between broader genome coverage versus maximal sequencing depth. We have validated reproducibility of the MPID assay in the detection of minority mutations in viral genomes. We have also examined the determinants that allow sequencing reads of PCR recombinants to contaminate the final TCS data set and show how such contamination can be limited. Finally, we provide several examples where we have applied MPID to analyze features of minority variants and describe limits on their detection in viral populations of HIV-1 and SARS-CoV-2 to demonstrate the generalizable utility of this approach with any RNA virus.

Technological and computational advances driving high-throughput oncology.

Engineering and computational advances have opened many new avenues in cancer research, particularly when being exploited in interdisciplinary approaches. For example, the combination of microfluidics, novel sequencing technologies, and computational analyses has been crucial to enable single-cell assays, giving a detailed picture of tumor heterogeneity for the very first time. In a similar way, these 'tech' disciplines have been elementary for generating large data sets in multidimensional cancer 'omics' approaches, cell-cell interaction screens, 3D tumor models, and tissue level analyses. In this review we summarize the most important technology and computational developments that have been or will be instrumental for transitioning classical cancer research to a large data-driven, high-throughput, high-content discipline across all biological scales.

Recent Developments in Mutation Enrichment and Detection Technologies.

Presence of excess unaltered, wild-type DNA (wtDNA) providing information of little clinical value may often mask low-level mutations containing important diagnostic or therapeutic clues. This is a recurring hurdle in biotechnology and medicine, including cancer, prenatal diagnosis, infectious diseases, and organ transplantation. Mutation enrichment techniques that allow reduction of unwanted DNA to enable the detection of low-level mutations have emerged since the early 1990s. They are continuously being refined and updated with new technologies. The burgeoning interest in liquid biopsies for residual cancer monitoring, detection of resistance to therapy, and early cancer detection has driven an expanded interest in new and improved methodologies for practical and effective mutation enrichment and detection of low-level mutations of clinical relevance. Newly developed mutation enrichment technologies are described and grouped according to the main principle of operation, PCR-blocking technologies, enzymatic methods, and physicochemical approaches. Special emphasis is given to technologies enabling pre-PCR blockage of wtDNA to bypass PCR errors [nuclease-assisted minor-allele enrichment assay with overlapping probes (NaME-PrO) and UV-mediated cross-linking minor allele enrichment (UVME)] or providing high multiplexity followed by next-generation sequencing [Minor allele enriched sequencing through recognition oligonucleotides (MAESTRO)]. This review summarizes technological developments in rare mutation enrichment over the last 12 years, complementing pre-2010 reviews on this topic. The expanding field of liquid biopsy calls for improved limits of detection (LOD) and highly parallel applications, along with the traditional requirements for accuracy, speed, and cost-effectiveness. The current technologies are reviewed with regards to these new requirements.

Mosaicism of common pathogenic MECP2 variants identified in two males with a clinical diagnosis of Rett syndrome.

Rett (RTT) syndrome, a neurodevelopmental disorder caused by pathogenic variation in the MECP2 gene, is characterized by developmental regression, loss of purposeful hand movements, stereotypic hand movements, abnormal gait, and loss of spoken language. Due to the X-linked inheritance pattern, RTT is typically limited to females. Recent studies revealed somatic mosaicism in MECP2 in male patients with RTT-like phenotypes. While detecting mosaic variation using Sanger sequencing is theoretically possible for mosaicism over ~15%-20%, several variables, including efficiency of PCR, background noise, and/or human error, contribute to a low detection rate using this technology. Mosaic variants in two males were detected by next generation sequencing (NGS; Case 1) and by Sanger re-sequencing (Case 2). Both had targeted digital PCR (dPCR) to confirm the variants. In this report, we present two males with classic RTT syndrome in whom we identified pathogenic variation in the MECP2 gene in the mosaic state (c.730C > T (p.Gln244*) in Patient 1 and c.397C > T (p.Arg133Cys) in Patient 2). In addition, estimates and measures of mosaic variant fraction were surprisingly similar between Sanger sequencing, NGS, and dPCR. The mosaic state of these variants contributed to a lengthy diagnostic odyssey for these patients. While NGS and even Sanger sequencing may be viable methods of detecting mosaic variation in DNA or RNA samples, applying targeted dPCR to supplement these sequencing technologies would provide confirmation of somatic mosaicism and mosaic fraction.

Targeted de novo phasing and long-range assembly by template mutagenesis.

Short-read sequencers provide highly accurate reads at very low cost. Unfortunately, short reads are often inadequate for important applications such as assembly in complex regions or phasing across distant heterozygous sites. In this study, we describe novel bench protocols and algorithms to obtain haplotype-phased sequence assemblies with ultra-low error for regions 10 kb and longer using short reads only. We accomplish this by imprinting each template strand from a target region with a dense and unique mutation pattern. The mutation process randomly and independently converts ∼50% of cytosines to uracils. Sequencing libraries are made from both mutated and unmutated templates. Using de Bruijn graphs and paired-end read information, we assemble each mutated template and use the unmutated library to correct the mutated bases. Templates are partitioned into two or more haplotypes, and the final haplotypes are assembled and corrected for residual template mutations and PCR errors. With sufficient template coverage, the final assemblies have per-base error rates below 10–9. We demonstrate this method on a four-member nuclear family, correctly assembling and phasing three genomic intervals, including the highly polymorphic HLA-B gene.

Pre-PCR Mutation-Enrichment Methods for Liquid Biopsy Applications.

Simple SummaryLiquid biopsies provide a non-invasive approach to tracing tumor-derived biomarkers in blood, with broad applications in medicine. During the past decade, circulating free DNA (cfDNA) has been turned into an informative resource for cancer management. Mutation-enrichment methods enhance the detection of tumor-derived, low-level mutations in blood. These methods increase the frequency of low-level mutations insofar as they become detectable via routine diagnostic techniques. Enriching mutations prior to PCR (pre-PCR) offers distinctive advantages. Applying the mutation-enrichment process directly to genomic DNA or cfDNA circumvents PCR errors and provides enriched mutation-containing products that different technologies can detect without any required changes in their protocols. In this review, we discuss the recent developments in pre-PCR enrichment methods from the perspective of their applications in liquid biopsies.Liquid biopsy is having a remarkable impact on healthcare- and disease-management in the context of personalized medicine. Circulating free DNA (cfDNA) is one of the most instructive liquid-biopsy-based biomarkers and harbors valuable information for diagnostic, predictive, and prognostic purposes. When it comes to cancer, circulating DNA from the tumor (ctDNA) has a wide range of applications, from early cancer detection to the early detection of relapse or drug resistance, and the tracking of the dynamic genomic make-up of tumor cells. However, the detection of ctDNA remains technically challenging, due, in part, to the low frequency of ctDNA among excessive circulating cfDNA originating from normal tissues. During the past three decades, mutation-enrichment methods have emerged to boost sensitivity and enable facile detection of low-level mutations. Although most developed techniques apply mutation enrichment during or following initial PCR, there are a few techniques that allow mutation selection prior to PCR, which provides advantages. Pre-PCR enrichment techniques can be directly applied to genomic DNA and diminish the influence of PCR errors that can take place during amplification. Moreover, they have the capability for high multiplexity and can be followed by established mutation detection and enrichment technologies without changes to their established procedures. The first approaches for pre-PCR enrichment were developed by employing restriction endonucleases directly on genomic DNA in the early 1990s. However, newly developed pre-PCR enrichment methods provide higher sensitivity and versatility. This review describes the available pre-PCR enrichment methods and focuses on the most recently developed techniques (NaME-PrO, UVME, and DEASH/MAESTRO), emphasizing their applications in liquid biopsies.

P606: META-ANALYSIS OF LOW ALLELIC BURDEN C481 BTK-MUTATIONS CALLS FOR CAUTION IN IBRUTINIB RESISTANCE EVALUATION

Background: Introduction of the orally administered tyrosine kinase inhibitor ibrutinib, a drug approved for relapsed or refractory mantle cell lymphoma in late 2013 and chronic lymphocytic leukemia in 2014, has changed treatment algoritms for B-cell malignancies and improved progression-free survival. Ibrutinib irreversibly targets Bruton’s tyrosine kinase (BTK), thus interrupting B-cell receptor signaling. However, it is known that mutations in the BTK gene, located on the X chromosome, can mediate acquired resistance, specifically modifying cysteine residue 481 to serine (C481S, X:101356176, GRCh38). Aims: Despite this knowledge, it is still unknown how low mutational burden can contribute to general resistance towards ibrutinib treatment. Thus, we performed a meta-analysis based on previous cardinal studies to investigate the collective and gender-specific distribution of variant allele frequencies. Methods: We implemented meta-analysis of single-nucleotide variant calls from seven studies involving whole-exome sequencing (Woyach et al. 2014 and 2017, Maddocks et al. 2015, Ahn et al. 2017, Kanagal-Shamanna et al. 2019, Gángó et al. 2020, Bödör et al. 2021) on mutational profiling concerning ibrutinib resistance and CLL. Four of these studies contained gender specific data. Computational analyses, statistics, and visualization were performed in Wolfram Language, R, and GraphPad Prism. Results: From the available data included in these studies, we were able to base our analyses on 226 non-BTK mutations and 170 BTK mutations within a variant allele frequency (VAF) range of 0.0001–0.9, of which 93 mutation candidates from males (VAF < 0.9) and 77 from females (VAF < 0.44) were observed. Extrapolative analysis of allele frequency distribution from all other variants (excl. BTK) in the studies revealed an exponential increase in the number of low burden variants below 10% VAF (R2>0.99). These variants were not significantly different between genders (PMann-Whitney=0.24, nmale=130, nfemale=73). In contrast, a significant difference was observed between male variant allele frequencies (PMann-Whitney=0.005) and females owing to BTK hemizygosity in males. However, this significance persisted when female BTK mutations were transformed to reflect a single copy (VAF/2, PMann-Whitney=0.015, ΔVAF=0.05) for gender comparison and even when observations for males were alternatively transformed to reflect two copies (2*VAF, PMann-Whitney<0.001, ΔVAF=0.19). The previously discovered threshold of 10% was set as the detection limit. Summary/Conclusion: Defining specific molecular contributions in drug resistance is imperative for the correct use of targeted therapy. It has been questioned how the low allelic burden of BTK mutations plays a role. Therefore, it is crucial to investigate the lower limit of detection. The meta-analysis suggests that variants below a certain threshold, here empirically 10%, may in many cases be false-positive variants, in agreement with other reports involving WES in general, introduced by erroneous base calling, PCR errors, etc. Although intriguing, it remains to elaborate whether the haploinsufficiency of BTK in males skew in the degree of ibrutinib resistance.

Maximum depth sequencing reveals an ON/OFF replication slippage switch and apparent in vivo selection for bifidobacterial pilus expression

The human gut microbiome, of which the genus Bifidobacterium is a prevalent and abundant member, is thought to sustain and enhance human health. Several surface-exposed structures, including so-called sortase-dependent pili, represent important bifidobacterial gut colonization factors. Here we show that expression of two sortase-dependent pilus clusters of the prototype Bifidobacterium breve UCC2003 depends on replication slippage at an intragenic G-tract, equivalents of which are present in various members of the Bifidobacterium genus. The nature and extent of this slippage is modulated by the host environment. Involvement of such sortase-dependent pilus clusters in microbe-host interactions, including bacterial attachment to the gut epithelial cells, has been shown previously and is corroborated here for one case. Using a Maximum Depth Sequencing strategy aimed at excluding PCR and sequencing errors introduced by DNA polymerase reagents, specific G-tract sequences in B. breve UCC2003 reveal a range of G-tract lengths whose plasticity within the population is functionally utilized. Interestingly, replication slippage is shown to be modulated under in vivo conditions in a murine model. This in vivo modulation causes an enrichment of a G-tract length which appears to allow biosynthesis of these sortase-dependent pili. This work provides the first example of productive replication slippage influenced by in vivo conditions. It highlights the potential for microdiversity generation in “beneficial” gut commensals.

Intra-genomic rRNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing.

SummaryRibosomal RNA (rRNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large‐scale exploration of environmental microbial diversity through metabarcoding approaches has been focused mainly on the V4 and V9 regions of the 18S rRNA gene. The accurate interpretation of such environmental surveys is hampered by technical (e.g. PCR and sequencing errors) and biological biases (e.g. intra‐genomic variability). Here we explored the intra‐genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra‐genomic variability of Nassellaria and Spumellaria is generally low, yet some Spumellaria specimens showed two different copies of the V4 with <97% similarity. Of the different sequencing methods, Illumina showed the highest number of contaminations (i.e. environmental DNA, cross‐contamination, tag‐jumping), revealed by its high sequencing depth; and MinION showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results highlight the requirement for a careful interpretation of Illumina‐based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full‐length rDNA environmental metabarcoding surveys.

Analysis of human BCR-IgH from genomic DNA with UMI incorporation

Abstract Immune repertoire analysis can be performed from practically any biological sample as long as viable nucleic acid can be extracted. While using RNA as a template has its benefits, gDNA is often preferred as gDNA is often easier to obtain, especially from difficult samples. In addition, since each cell may only have one copy of the successfully rearranged V(D)J, it may better reflect the quantity of cells. In other words, identification of a successfully rearranged V(D)J will not be skewed by expression levels as the relationship should be one cell to one V(D)J rearrangement. We developed a new set of BCR-IgH specific primers to target the gDNA V(D)J region while also incorporating unique molecular indices (UMI) through our proprietary dimer avoided multiplex PCR technology. The inclusion of UMI with gDNA amplification allows each cell to be counted since the relationship of one V(D)J per cell will be maintained and will have a unique barcode added during the processing. The inclusion of UMI also assists with the elimination of PCR and sequencing error, which will lead to more confident analysis of low frequency clones and hypermutations. The primers were first tested on a BCR-IgH synthetic library. The sequencing result demonstrated &amp;gt;85% correlation with the known synthetic library gene expression. These results showed that the new gDNA BCR-IgH panel was sufficient at amplifying all known J gene targets. Presently, we have also applied this method to gDNA extracted from B cell enriched PBMC samples including the spike-in of a known BCR clone at various concentrations. To our knowledge, this is the first assay of its kind to incorporate UMI into BCR-IgH gDNA to capture both the BCR repertoire and true cell frequency.