Dual Indexing Research Articles

Measuring B Cell Chromatin Accessibility by One-Step ATAC-seq.

The Assay for Transposase Accessible Chromatin (ATAC)-seq protocol is optimized to generate global maps of accessible chromatin using limited cell inputs. The Tn5 transposase tagmentation reaction simultaneously fragments and tags the accessible DNA with Illumina Nextera sequencing adapters. Fragmented and adapter tagged DNA is then purified and PCR amplified with dual indexing primers to generate a size-specific sequencing library. The One-Step workflow below outlines the Tn5 nuclei transposition from a range of cell inputs followed by PCR amplification to generate a sequencing library.

PetaKV: Building Efficient Key-Value Store for File System Metadata on Persistent Memory

Previous works proposed building file systems and organizing the metadata with KV stores because KV stores handle entries of various sizes efficiently and have excellent scalability. The emergence of the byte-addressable persistent memory (PM) enables metadata service to be faster than before by tailoring the KV store for the PM. However, existing PM-based KV stores cannot handle the workloads of file systems’ metadata well because simply depending on hash tables or trees cannot simultaneously provide fast file accessing and efficient directory traversing. In this paper, we exploit the insight of the metadata operations and propose the PetaKV, a KV store tailored for the metadata management of file systems on PM. PetaKV leverages dual hash indexing to achieve fast file put and get operations. Moreover, it cooperates with PM-tailored peta logs to collocate KV entries for each directory, thus supporting efficient directory scans. Our evaluation indicates PetaKV outperforms state-of-art tree-based KV stores on put, get and scan <inline-formula><tex-math notation="LaTeX">$2.5\times$</tex-math></inline-formula> , <inline-formula><tex-math notation="LaTeX">$3.2\times$</tex-math></inline-formula> , and <inline-formula><tex-math notation="LaTeX">$2.8\times$</tex-math></inline-formula> on average, respectively. Moreover, the file system built with PetaKV achieves <inline-formula><tex-math notation="LaTeX">$1.2\times$</tex-math></inline-formula> to <inline-formula><tex-math notation="LaTeX">$6.4\times$</tex-math></inline-formula> speedup compared to those built with tree-based KV stores on the metadata operations.

DAMNet: A Dual Adjacent Indexing and Multi-Deraining Network for Real-Time Image Deraining

Image deraining is increasingly critical in the domain of computer vision. However, there is a lack of fast deraining algorithms for multiple images without temporal and spatial features. To fill this gap, an efficient image-deraining algorithm based on dual adjacent indexing and multi-deraining layers is proposed to increase deraining efficiency. The deraining operation is based on two proposals: the dual adjacent method and the joint training method based on multi-deraining layers. The dual adjacent structure indexes pixels from adjacent features of the previous layer to merge with features produced by deraining layers, and the merged features are reshaped to prepare for the loss computation. Joint training method is based on multi-deraining layers, which utilise the pixelshuffle operation to prepare various deraining features for the multi-loss functions. Multi-loss functions jointly compute the structural similarity by loss calculation based on reshaped and deraining features. The features produced by the four deraining layers are concatenated in the channel dimension to obtain the total structural similarity and mean square error. During the experiments, the proposed deraining model is relatively efficient in primary rain datasets, reaching more than 200 fps, and maintains relatively impressive results in single and crossing datasets, demonstrating that our deraining model reaches one of the most advanced ranks in the domain of rain-removing.

324. Detection of Antibiotic Resistance Genes and Associated Genera Direct from Blood Samples by Multiplex PCR and Next Generation Sequencing

Abstract Background While blood culture and genotypic or phenotypic testing is standard procedure for identification of sepsis pathogens and resistance, blood culture negative sepsis occurs in 40-60% of patients. A culture independent diagnostic can identify pathogens that do not grow in culture and may provide a more sensitive indicator to blood stream infections. Here we discuss an assay that combines multiplex PCR with next generation sequencing (NGS) to detect resistance genes and pathogenic genera directly from whole blood. These genes include mecA, mecC, vanA, vanB, blaKPC, blaOXA-48 family, blaNDM, blaVIM, blaIMP, blaCMY-2, blaDHA, and blaCTX-M-14 and blaCTX-M-15 groups. Methods K2EDTA whole blood samples were spiked with low titers of antimicrobial resistant bacterial isolates. Sample processing involved chemical lysis of red blood cells, concentration and mechanical lysis of pathogen cells, and multiplex amplification of resistance genes and 16S rRNA gene. A synthetic DNA oligo was added at a fixed concentration as a read control. Samples were sequenced on Illumina platforms. NGS data was analyzed using a bioinformatics pipeline to process and match sequences to curated databases with BLAST. The number of reads per sequence was normalized by the number of reads for the read control, resulting in a read-control-normalized (RCN) value that could be used to establish positivity cutoffs. Results Resistance genes in bacterial pathogen spiked samples were identified at titers ≤ 10 CFU/mL. Owing to a unique dual indexing strategy, cross-reactivity was absent or low enough that cutoffs could be set using RCN values without reducing sensitivity. The expected genus was also detected at ≤ 10 CFU/mL for all samples; however, genera that may be representative of reagent or environmental contamination were observed at levels higher than a universal RCN cutoff in some samples. Conclusion We have shown that this NGS assay can detect 13 antimicrobial resistance genes with high sensitivity and specificity direct from blood. Bacterial genera associated with these genes were detected by this assay. As there is a risk of detection of contaminating species with a 16S target, further research into low DNA reagents or environmental controls may be required. Disclosures Daniel Gamero, BS, T2 Biosystems, inc: WO2018213641A8, EP3861013A1, WO2020055887A1, WO2020252084A1, WO2020257691A1|T2 Biosystems, inc: Stocks/Bonds Kelly Dabrowski, M.S., T2 Biosystems, Inc: Stocks/Bonds Analisa DeVito, B.S., T2 Biosystems, Inc: Stocks/Bonds Roger Smith, Ph.D., T2 Biosystems, Inc: US20180188195A1, EP3849938A1,WO2020257691A1, WO2020252084A1|T2 Biosystems, Inc: Stocks/Bonds Jessica L. Snyder, Ph.D., T2 Biosystems, Inc: CA3062882A1,US20180171388A1,WO2017180745A1 , EP3861013A1 , AU2020296183A1, WO2020055887A1, WO2020252084A1|T2 Biosystems, Inc: Stocks/Bonds.

High-throughput barcoding method for the genetic surveillance of insecticide resistance and species identification in Anopheles gambiae complex malaria vectors

Surveillance of malaria vector species and the monitoring of insecticide resistance are essential to inform malaria control strategies and support the reduction of infections and disease. Genetic barcoding of mosquitoes is a useful tool to assist the high-throughput surveillance of insecticide resistance, discriminate between sibling species and to detect the presence of Plasmodium infections. In this study, we combined multiplex PCR, custom designed dual indexing, and Illumina next generation sequencing for high throughput single nucleotide polymorphism (SNP)-profiling of four species from the Anopheles (An.) gambiae complex (An. gambiae sensu stricto, An. coluzzii, An. arabiensis and An. melas). By amplifying and sequencing only 14 genetic fragments (500 bp each), we were able to simultaneously detect Plasmodium infection; insecticide resistance-conferring SNPs in ace1, gste2, vgsc and rdl genes; the partial sequences of nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and intergenic spacers (IGS), Short INterspersed Elements (SINE), as well as mitochondrial genes (cox1 and nd4) for species identification and genetic diversity. Using this amplicon sequencing approach with the four selected An. gambiae complex species, we identified a total of 15 non-synonymous mutations in the insecticide target genes, including previously described mutations associated with resistance and two new mutations (F1525L in vgsc and D148E in gste2). Overall, we present a reliable and cost-effective high-throughput panel for surveillance of An. gambiae complex mosquitoes in malaria endemic regions.

Implementation of GA-VirReport, a Web-Based Bioinformatics Toolkit for Post-Entry Quarantine Screening of Virus and Viroids in Plants.

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21–22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

Fecal microbiota transplantation capsule therapy via oral route for combatting atopic dermatitis in dogs

Given the role of the interaction between gut microbiome with dermatological diseases, namely “gut-skin axis”, the present author proved that gut restoration should alleviate canine atopic dermatitis (CAD), which was the purpose of the study. A 4-week, open-label, non-controlled case series involved 8 -owned dogs with CAD which had received no previous treatment. Evaluations included Canine Atopic Dermatitis Extent and Severity Index version 4 (CADESI-04 scores), Visual Analog Scale (VAS) pruritus scores and Polycheck in vitro allergen specific tests. Faecal samples were analysed by dual indexing one-step PCR and 16S rRNA targeted metagenomics for detecting gut microbiota alterations before and after fecal microbiota transplantation (FMT) capsule treatment twice daily for 4 weeks. All cases were presenting pruritus and all of those dogs showed elevated IgE levels. CADESI scores decreased on days 28 (4-21) compared to day 0 initial values (50-128). Similarly, decreased VAS scores were detected on days 28 (0-2) in contrast to prior values (6-10). Regarding epidermal barrier functioning epidermal hydration (55-100 vs. 4-24) and pH (6.-7.8 vs. 4.2-5.7) values were elevated after FMT treatment in contrast to prior ranges, respectively. Alpha diversity revaled both richness and diversity of gut microbiota were improved for all cases on day 28. Furthermore at the end of trial Firmicutes: Bacteroidetes ratio was 8, the benchmark detected for healthy dogs. The present study supports a potential benefit of FMT capsule treatment against CAD. This safe and tolerant treatment modality directed against CAD shifted the gut microbiome composition towards a healthy state for all 8 dogs enrolled.

Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

Abstract PS2-05: Genetic profiling for circulating tumor cell clusters to unveil molecular drivers of metastasis

Abstract Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. A portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in MBC as compared to individual CTCs (Aceto N. Cell, 2015). However, genomic characterization of CTCs-clusters compared to single CTCs remain largely unknown. We previously reported single CTC sequencing for HER2+ CTCs (2020 AACR #3120). Herein, we report a new finding of heterogeneity profiling for CTC-clusters compared to single CTCs, which would be helpful to evaluate the MBC metastasis capability and treatment in clinic. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration was performed using the FDA-cleared CellSearch™ System (Menarini) targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The CTC-clusters and single CTCs were isolated using DEPArrayTM System (Menarini). DNA was isolated from CTC-clusters and single CTCs by ArcturusTM PicoPureTM DNA Extraction kit. The initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing. Results: We identified 107 CTCs by CellSearch™, including 93 single CTCs, 14 CTC-clusters and 145 WBCs. Autologous CTC-clusters (CK+DAPI+CD45-, Group 1), single CTCs (CK+DAPI+CD45-, Group 2), and leukocytes (CK-DAPI+CD45+, Group 3) were sequenced respectively. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 60,638 counts (6.77%) and 70,334 counts (8.20%) for exon variants in CTC-clusters and single CTCs respectively, 507,595 counts (56.69%) and 486,119 counts (56.69%) for intron variants, 194,026 (21.67%) and 175,819 counts (20.51%) for intergenic variants, 54,174 counts (6.05%) and 50,370 counts (5.87%) for downstream genes, 51,716 counts (5.78%) and 45,915 counts (5.36%) for upstream genes, and 3.04% and 3.37% of others variants in CTC-clusters and single CTCs respectively. Meanwhile, there was 0 count for exon and intron variants found in Group 3. There were 60 and 79 gene variants (SNP and Ins-Del) identified to have the highest impact effect (≥20) on CTC-clusters and single CTC exons respectively, which affect significantly on the functional proteins coding. Among the top 50 high impact gene variants in each group, there were 25 gene alteration sites were similar in Group 1 and 2, including XYLB, RAN, QPCT, HPGDS, HDAC8, GABBR2, CYP11B2 and CHKA. Specific to Group 1, there were 25 gene alterations which were primarily related to cellular proliferation and tumor promotion (AMD1), liver drug clearance (CES1), tissue remodeling (CHI3L1), immune cytokine signaling (JAK1) and metabolism (ASRGL1). Meanwhile, there are 25 specific gene alterations in Group 2 compared to Group 1, which were associated with nucleotide-excision repair (DDB1 and FAN1) chromosome positioning (KIF11), cell growth, differentiation, mitotic cycle, oncogenic transformation (PTPN3 and MAPK14), apoptosis (CASP1) and cell growth (CTNNB1). Conclusion: Genomic characterization of CTC-clusters compared to autologous single CTCs and leukocytes elucidated new specific gene alterations in CTC-clusters associated with most aggressive disease metastasis in MBC, which will help to gain new insights on the molecular mechanisms associated with the metastasis and find new molecularly driven therapies for disease metastasis. Citation Format: Qiang Zhang, Lorenzo Gerratana, Paolo D'Amico, Andrew A. Davis, Saya Liz Jacob, Xinkun Wang, Zhe Ji, Zheng Cai, Elena Vagia, Wenan Qiang, Ami Shah, Youbin Zhang, Lisa Flaum, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic profiling for circulating tumor cell clusters to unveil molecular drivers of metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-05.

Abstract 3120: Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing

Abstract Introduction: Although CTCs display the same spatial and temporal heterogeneity as the primary tumor, they represent a privileged window to disclose mechanisms of metastases. We previously reported that overexpression of HER2 in CTCs is associated with worse prognosis in MBC (2018 AACR #5195). Herein, we report a new finding of heterogeneity profiling for HER2 positive CTC by using a new approach for single CTC isolation and sequencing. Methods: Whole blood sample (7.5ml/each) was collected from stage III/IV MBC patients before therapy. CTC enumeration were performed in FDA approved CellSearch™ System by targeting the EpCAM antigen for capturing CTCs which were then stained by Anti-CK-PE, DAPI, anti-CD45-APC and anti-HER2-FITC. The single HER2+ CTCs were isolated by using DEPArrayTM System (Menarini). The single cell DNA was isolated and the initial library was prepared by SMARTer® PicoPLEX® Gold Single Cell DNA-Seq Kit, and the exome capture was performed by Twist Human Core Exome EF Multiplex Complete Kit. The sequencing was prepared by NextSeq 500 mid output V2.5 kit and was performed on the NextSeq 500 (Illumina). It was a paired end run, 75×75 bps run with dual indexing. Results: We identified 208 CTCs by CellSearch™ system, including 114 HER2+ CTCs and 4 CTC-clusters. Single HER2+ CTC was isolated and sequenced. The sequencing data was processed following the GATK pipeline and annotated using SnpEff. There were 362,015 counts (58.23%) for intron variants, 104,011 counts (16.73%) for intergenic variants, 56,678 counts (9.19%) for exon variants, 41,133 counts (6.67%) for downstream genes, 33,903 counts (5.45%) for upstream genes and others (3.73%). There were 44 genes (64 sites) variants were identified to have highest impact effect (≥20) on HER2+ CTC chromosome, among which TP53, PIK3CA, ESR1, PIK3R1 variants matched the most recent genomic characterization report for MBC tissue (Bertucci F. Nature 2019) when the XYLB, CYP17A1, CA2 and CAD variants were firstly reported. More important, although overexpression of PTP1B (PTPN1) was implicated in the development of MBC, our evidences firstly demonstrated that PTPN1 mutation is the most important variant (≥20) in CTCs for MBC. Furthermore, we also found that ESR1 mutation in CTC correlated with ESR1 status in ctDNA. Except for the high impact variants, there were 42 genes (66 sites) and 83 genes (136 sites) have medium (10-19) and significant (5-9) impact variants respectively. Conclusion: Genomic characterization of HER2 positive single CTCs will elucidate new specific gene alterations (e.g. PTPN1) associated with disease metastasis, which will improve understanding of this critical process in MBC and lead to the development of novel drugs aimed at their eradication using molecularly driven therapies for this deadly condition. Citation Format: Qiang Zhang, Lorenzo Gerratana, Kara L. Pivarski, Xinkun Wang, Zhe Ji, Emily Stroup, Elena Vagia Vagia, Andrew Adam Davis A. Adam Davis, Ami Shah, Katy L. Kerby, Lisa Flaum Flaum, Firas Wehbe, Youbin Zhang, Wenan Qiang, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. Genetic heterogeneity profiling for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application of DEPArrayTM System and single cell DNA sequencing for HER2 positive single circulating tumor cell (CTC) in metastatic breast cancer (MBC) by application DEPArrayTM system and single cell DNA sequencing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3120.

Unique dual indexing PCR reduces chimeric contamination and improves mutation detection in cell-free DNA of pregnant women

Allele fraction measurement is an essential component in nucleic acid analysis. The formation of chimeric amplicons during multiplex PCR amplification, however, greatly affects the allele fraction even before downstream analysis. Previous error correction strategy with unique molecular indexing (UMI) targets mainly points mutations rather than chimeras. Since the mutant allele detection in pregnant women cell-free DNA (cfDNA) is limited by chimeric amplicon contamination, a more direct error correction solution is demanded. Here we demonstrate effective reduction of chimeric amplicon contamination by unique dual indexing. With error corrected deep sequencing analysis, we achieved 100% accuracy in 16 tests of the parental mutation inheritance and de novo mutations in cfDNA of pregnant women, whose fetuses were at risk of tuberous sclerosis complex or Marfan syndrome. Our error correction strategy could offer a versatile solution for accurate multiplex PCR amplification.

Meta-Prism: Ultra-fast and highly accurate microbial community structure search utilizing dual indexing and parallel computation.

Microbiome samples are accumulating at an unprecedented speed. As a result, a massive amount of samples have become available for the mining of the intrinsic patterns among them. However, due to the lack of advanced computational tools, fast yet accurate comparisons and searches among thousands to millions of samples are still in urgent need. In this work, we proposed the Meta-Prism method for comparing and searching the microbial community structures amongst tens of thousands of samples. Meta-Prism is at least 10 times faster than contemporary methods serving the same purpose and can provide very accurate search results. The method is based on three computational techniques: dual-indexing approach for sample subgrouping, refined scoring function that could scrutinize the minute differences among samples, and parallel computation on CPU or GPU. The superiority of Meta-Prism on speed and accuracy for multiple sample searches is proven based on searching against ten thousand samples derived from both human and environments. Therefore, Meta-Prism could facilitate similarity search and in-depth understanding among massive number of heterogenous samples in the microbiome universe. The codes of Meta-Prism are available at: https://github.com/HUST-NingKang-Lab/metaPrism.

3\u2019Pool-seq: an optimized cost-efficient and scalable method of whole-transcriptome gene expression profiling

BackgroundThe advent of Next Generation Sequencing has allowed transcriptomes to be profiled with unprecedented accuracy, but the high costs of full-length mRNA sequencing have posed a limit on the accessibility and scalability of the technology. To address this, we developed 3’Pool-seq: a simple, cost-effective, and scalable RNA-seq method that focuses sequencing to the 3′-end of mRNA. We drew from aspects of SMART-seq, Drop-seq, and TruSeq to implement an easy workflow, and optimized parameters such as input RNA concentrations, tagmentation conditions, and read depth specifically for bulk-RNA.ResultsThorough optimization resulted in a protocol that takes less than 12 h to perform, does not require custom sequencing primers or instrumentation, and cuts over 90% of the costs associated with TruSeq, while still achieving accurate gene expression quantification (Pearson’s correlation coefficient with ERCC theoretical concentration r = 0.96) and differential gene detection (ROC analysis of 3’Pool-seq compared to TruSeq AUC = 0.921). The 3’Pool-seq dual indexing scheme was further adapted for a 96-well plate format, and ERCC spike-ins were used to correct for potential row or column pooling effects. Transcriptional profiling of troglitazone and pioglitazone treatments at multiple doses and time points in HepG2 cells was then used to show how 3’Pool-seq could distinguish the two molecules based on their molecular signatures.Conclusions3’Pool-seq can accurately detect gene expression at a level that is on par with TruSeq, at one tenth of the total cost. Furthermore, its unprecedented TruSeq/Nextera hybrid indexing scheme and streamlined workflow can be applied in several different formats, including 96-well plates, which allows users to thoroughly evaluate biological systems under several conditions and timepoints. Care must be taken regarding experimental design and plate layout such that potential pooling effects can be accounted for and corrected. Lastly, further studies using multiple sets of ERCC spike-ins may be used to simulate differential gene expression in a system with known ground-state values.

Proof of concept: used malaria rapid diagnostic tests applied for parallel sequencing for surveillance of molecular markers of anti-malarial resistance in Bissau, Guinea-Bissau during 2014\u20132017

BackgroundLarge-scale surveillance of molecular markers of anti-malarial drug resistance is an attractive method of resistance monitoring, to complement therapeutic efficacy studies in settings where the latter are logistically challenging.MethodsBetween 2014 and 2017, this study sampled malaria rapid diagnostic tests (RDTs), used in routine clinical care, from two health centres in Bissau, Guinea-Bissau. In order to obtain epidemiological insights, RDTs were collected together with patient data on age and sex. A subset of positive RDTs from one of the two sites (n = 2184) were tested for Plasmodium DNA content. Those testing positive for Plasmodium DNA by PCR (n = 1390) were used for library preparation, custom designed dual indexing and next generation Miseq targeted sequencing of Plasmodium falciparum genes pfcrt, pfmdr1, pfdhfr, pfdhps and pfk13.ResultsThe study found a high frequency of the pfmdr1 codon 86N at 88–97%, a significant decrease of the pfcrt wildtype CVMNK haplotype and elevated levels of the pfdhfr/pfdhps quadruple mutant ranging from 33 to 51% between 2014 and 2017. No polymorphisms indicating artemisinin tolerance were discovered. The demographic data indicate a large proportion of young adults (66%, interquartile range 11–28 years) presenting with P. falciparum infections. While a total of 5532 gene fragments were successfully analysed on a single Illumina Miseq flow cell, PCR-positivity from the library preparation varied considerably from 13 to 87% for different amplicons. Furthermore, pre-screening of samples for Plasmodium DNA content proved necessary prior to library preparation.ConclusionsThis study serves as a proof of concept for using leftover clinical material (used RDTs) for large-scale molecular surveillance, encompassing the inherent complications regarding to methodology and analysis when doing so. Factors such as RDT storage prior to DNA extraction and parasitaemia of the infection are likely to have an effect on whether or not parasite DNA can be successfully analysed, and are considered part of the reason the data yield is suboptimal. However, given the necessity of molecular surveillance of anti-malarial resistance in settings where poor infrastructure, poor economy, lack of educated staff and even surges of political instability remain major obstacles to performing clinical studies, obtaining the necessary data from used RDTs, despite suboptimal output, becomes a feasible, affordable and hence a justifiable method.

Abstract 3526: Incorporation of unique molecular identifiers (UMIs) into unique dual sample indexing (UDI) improves the accuracy of quantitative next generation sequencing

Abstract Accurate analysis of quantitative NGS data is critical for low frequency variant detection, identification of differentially expressed transcripts, and correct diagnosis and patient care in a clinical NGS setting. Two major factors affecting sequencing accuracy are 1) PCR duplication arising from amplification of library molecules; and 2) errors introduced during library preparation and actual sequencing on the flow cell. Standard practice for identification and removal of PCR duplicates relies on aligning reads to the same genomic coordinates. However, this approach can not differentiate between PCR duplicates and reads originating from unique molecules with identical ends. The most effective method for error correction is Duplex Sequencing, which utilizes UMIs to tag both strands of each individual DNA duplex followed by building consensus sequences. Unfortunately, this approach is labor intensive and cost-prohibitive for complex genomes and large target panels. Therefore, a simple and reliable approach for duplicate removal and error correction would greatly facilitate the wider adoption of NGS technology for diagnostics and clinical applications. In this study, we incorporate UMIs into UDI systems and assess the effect on the accuracy of quantitative sequencing assays. We first study the effectiveness of various computational methods to account for UMIs and remove base-calling errors introduced during sequencing. We then analyze the utility of UMIs for 1) unique and low abundance transcript identification and accurate transcript quantification; and 2) error correction in low frequency variant detection in genomic sequencing from both high quality cell line DNA and low quality FFPE DNA. In addition, we demonstrate that combining unique dual sample indexing with UMI molecular barcoding further improves data analysis accuracy, especially on patterned flow cells. Our approach involves a simple new UMI-containing UDI adaptor design that can also be applied to other sequencing methods and platforms. Citation Format: Pingfang Liu, Keerthana Krishnan, Camille X. Devoe, Bradley W. Langhorst, Eileen Dimalanta, Theodore B. Davis. Incorporation of unique molecular identifiers (UMIs) into unique dual sample indexing (UDI) improves the accuracy of quantitative next generation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3526.

Towards an in-depth characterization of Symbiodiniaceae in tropical giant clams via metabarcoding of pooled multi-gene amplicons.

High-throughput sequencing is revolutionizing our ability to comprehensively characterize free-living and symbiotic Symbiodiniaceae, a diverse dinoflagellate group that plays a critical role in coral reef ecosystems. Most studies however, focus on a single marker for metabarcoding Symbiodiniaceae, potentially missing important ecological traits that a combination of markers may capture. In this proof-of-concept study, we used a small set of symbiotic giant clam (Tridacna maxima) samples obtained from nine French Polynesian locations and tested a dual-index sequence library preparation method that pools and simultaneously sequences multiple Symbiodiniaceae gene amplicons per sample for in-depth biodiversity assessments. The rationale for this approach was to allow the metabarcoding of multiple genes without extra costs associated with additional single amplicon dual indexing and library preparations. Our results showed that the technique effectively recovered very similar proportions of sequence reads and dominant Symbiodiniaceae clades among the three pooled gene amplicons investigated per sample, and captured varying levels of phylogenetic resolution enabling a more comprehensive assessment of the diversity present. The pooled Symbiodiniaceae multi-gene metabarcoding approach described here is readily scalable, offering considerable analytical cost savings while providing sufficient phylogenetic information and sequence coverage.

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons.

To improve our knowledge on the epidemiological status of African trypanosomiasis, better tools are required to monitor Trypanosome genotypes circulating in both mammalian hosts and tsetse fly vectors. This is important in determining the diversity of Trypanosomes and understanding how environmental factors and control efforts affect Trypanosome evolution. We present a single test approach for molecular detection of different Trypanosome species and subspecies using newly designed primers to amplify the Internal Transcribed Spacer 1 region of ribosomal RNA genes, coupled to Illumina sequencing of the amplicons. The protocol is based on Illumina’s widely used 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. Results from analysis of wild tsetse flies collected from Zambia and Zimbabwe show that conventional methods for Trypanosome species detection based on band size comparisons on gels is not always able to accurately distinguish between T. vivax and T. godfreyi. Additionally, this approach shows increased sensitivity in the detection of Trypanosomes at species level with the exception of the Trypanozoon subgenus. We identified subspecies of T. congolense, T. simiae, T. vivax, and T. godfreyi without the need for additional tests. Results show T. congolense Kilifi subspecies is more closely related to T. simiae than to other T. congolense subspecies. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any subspecies for T. godfreyi, we observed two distinct clusters for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from other T. simiae Tsavo sequences suggesting the Nannomonas group is more divergent than currently thought thus the need for better classification criteria. This method presents a simple but comprehensive way of identification of Trypanosome species and subspecies-specific using one PCR assay for molecular epidemiology of trypanosomes.

Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms

BackgroundHere we present an in-depth characterization of the mechanism of sequencer-induced sample contamination due to the phenomenon of index swapping that impacts Illumina sequencers employing patterned flow cells with Exclusion Amplification (ExAmp) chemistry (HiSeqX, HiSeq4000, and NovaSeq). We also present a remediation method that minimizes the impact of such swaps.ResultsLeveraging data collected over a two-year period, we demonstrate the widespread prevalence of index swapping in patterned flow cell data. We calculate mean swap rates across multiple sample preparation methods and sequencer models, demonstrating that different library methods can have vastly different swapping rates and that even non-ExAmp chemistry instruments display trace levels of index swapping. We provide methods for eliminating sample data cross contamination by utilizing non-redundant dual indexing for complete filtering of index swapped reads, and share the sequences for 96 non-combinatorial dual indexes we have validated across various library preparation methods and sequencer models. Finally, using computational methods we provide a greater insight into the mechanism of index swapping.ConclusionsIndex swapping in pooled libraries is a prevalent phenomenon that we observe at a rate of 0.2 to 6% in all sequencing runs on HiSeqX, HiSeq 4000/3000, and NovaSeq. Utilizing non-redundant dual indexing allows for the removal (flagging/filtering) of these swapped reads and eliminates swapping induced sample contamination, which is critical for sensitive applications such as RNA-seq, single cell, blood biopsy using circulating tumor DNA, or clinical sequencing.

Preparation of highly multiplexed small RNA sequencing libraries.

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology

Genetic polymorphisms in P. falciparum can be used to indicate the parasite’s susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8–100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.