Standard Illumina Research Articles

Benchmarking of ATAC Sequencing Data From BGI's Low-Cost DNBSEQ-G400 Instrument for Identification of Open and Occupied Chromatin Regions.

Background: Chromatin falls into one of two major subtypes: closed heterochromatin and euchromatin which is accessible, transcriptionally active, and occupied by transcription factors (TFs). The most widely used approach to interrogate differences in the chromatin state landscape is the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). While library generation is relatively inexpensive, sequencing depth requirements can make this assay cost-prohibitive for some laboratories. Findings: Here, we benchmark data from Beijing Genomics Institute’s (BGI) DNBSEQ-G400 low-cost sequencer against data from a standard Illumina instrument (HiSeqX10). For comparisons, the same bulk ATAC-seq libraries generated from pluripotent stem cells (PSCs) and fibroblasts were sequenced on both platforms. Both instruments generate sequencing reads with comparable mapping rates and genomic context. However, DNBSEQ-G400 data contained a significantly higher number of small, sub-nucleosomal reads (>30% increase) and a reduced number of bi-nucleosomal reads (>75% decrease), which resulted in narrower peak bases and improved peak calling, enabling the identification of 4% more differentially accessible regions between PSCs and fibroblasts. The ability to identify master TFs that underpin the PSC state relative to fibroblasts (via HOMER, HINT-ATAC, TOBIAS), namely, foot-printing capacity, were highly similar between data generated on both platforms. Integrative analysis with transcriptional data equally enabled direct recovery of three published 3-factor combinations that have been shown to induce pluripotency. Conclusion: Other than a small increase in peak calling sensitivity for DNBSEQ-G400 data (BGI), both platforms enable comparable levels of open chromatin identification for ATAC-seq library sequencing, yielding similar analytical outcomes, albeit at low-data generation costs in the case of the BGI instrument.

A Rapid, Whole Genome Sequencing Assay for Detection and Characterization of Novel Coronavirus (SARS-CoV-2) Clinical Specimens Using Nanopore Sequencing.

A new human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged at the end of 2019 in Wuhan, China that caused a range of disease severities; including fever, shortness of breath, and coughing. This disease, now known as coronavirus disease 2019 (COVID-19), quickly spread throughout the world, and was declared a pandemic by the World Health Organization in March of 2020. As the disease continues to spread, providing rapid characterization has proven crucial to better inform the design and execution of control measures, such as decontamination methods, diagnostic tests, antiviral drugs, and prophylactic vaccines for long-term control. Our work at the United States Army’s Combat Capabilities Development Command Chemical Biological Center (DEVCOM CBC) is focused on engineering workflows to efficiently identify, characterize, and evaluate the threat level of any potential biological threat in the field and more remote, lower resource settings, such as forward operating bases. While we have successfully established untargeted sequencing approaches for detection of pathogens for rapid identification, our current work entails a more in-depth sequencing analysis for use in evolutionary monitoring. We are developing and validating a SARS-CoV-2 nanopore sequencing assay, based on the ARTIC protocol. The standard ARTIC, Illumina, and nanopore sequencing protocols for SARS-CoV-2 are elaborate and time consuming. The new protocol integrates Oxford Nanopore Technology’s Rapid Sequencing Kit following targeted RT-PCR of RNA extracted from human clinical specimens. This approach decreases sample manipulations and preparation times. Our current bioinformatics pipeline utilizes Centrifuge as the classifier for quick identification of SARS-CoV-2 and RAMPART software for verification and mapping of reads to the full SARS-CoV-2 genome. ARTIC rapid sequencing results, of previous RT-PCR confirmed patient samples, showed that the modified protocol produces high quality data, with up to 98.9% genome coverage at >1,000x depth for samples with presumably higher viral loads. Furthermore, whole genome assembly and subsequent mutational analysis of six of these sequences identified existing and unique mutations to this cluster, including three in the Spike protein: V308L, P521R, and D614G. This work suggests that an accessible, portable, and relatively fast sample-to-sequence process to characterize viral outbreaks is feasible and effective.

MO374: Urinary Cell Transcriptomics Provides a Non-Invasive Readout of Kidney Genes Essential to Renal Health and Disease

Abstract BACKGROUND AND AIMS Chronic kidney disease affects up to 15% of adults, current diagnostic methods for assessing kidney health and early detection of renal disease lack sensitivity and/or can be highly invasive. The kidney sheds cells into the urine and harvesting these cells and their transcriptomic profile could yield non-invasive insights into genes responsible for maintenance of kidney health and early detection of kidney disease. METHOD Cell pellets were isolated from 200 mL of fresh morning urine from 33 participants, samples were spun at 3000 g for 10 min, washed and then resuspended in PBS. Each urinary cell pellet was then profiled by standard Illumina poly-A RNA-sequencing, generating an average of 30 million paired reads per sample. These were quantified using the standard GTEx quantification pipeline and were compared against 43 different human tissues and other bodily fluids. RESULTS Detailed RNA-sequencing metric analysis revealed that urinary cell pellets can generate reliable data of comparable or better quality than saliva and cerebrospinal fluid at similar read coverage. The one hundred most highly expressed urinary cell genes show an enrichment for immunity (P = 1.2 × 10–7), glucose metabolism (P = 1.3 × 10–5) and renal mineral absorption (P = 9.8 × 10–4); themes shared with the renal transcriptome. Across all protein-coding genes, kidney cortex (r2 = 0.65) and kidney medulla (r2 = 0.64) showed the highest level of correlation with the urinary transcriptome in an analysis of 43 human tissues. The correlation between urinary cells and the kidney was particularly strong (r2 = 0.72) in an analysis restricted to highly specific kidney genes (including UMOD, KCNJ1 and SLC12A1 all known for their role in kidney health and disease). CONCLUSION Poly-A RNA-sequencing of cells harvested from human urine can yield high quality gene expression profiles that correlate with transcriptional activity of the kidney. RNA-sequencing-based profiling of urinary cells offers a completely non-invasive route to assessing the expression of kidney genes of key relevance to renal health and disease.

Abstract P2-06-04: Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers

Abstract Background: Cancer genomes provide a durable record of the genetic alterations that are acquired during normal cell development and carcinogenesis from DNA damage and DNA repair defects. As DNA repair-deficient tumors often become dependent on backup repair pathways, mutational signatures found in such tumors are thought to reflect the absence of a particular repair pathway as well as the activity of the backup repair mechanism responsible for maintaining genome integrity. While homologous recombination (HR) deficiency is primarily a disorder of double-strand break (DSB) repair, the mutation classes most specifically associated with HR-deficiency in cancer are paradoxically small variants, namely single nucleotide variants and short deletions. Mechanistic and cytogenetic studies, however, indicate that HR-deficiency should compromise structural genomic integrity and yield complex rearrangements. Here we elucidate complex structural variants that are specific for HR-deficient cancers and identify rearrangements that differentiate BRCA1 from BRCA2 loss and illuminate divergent backup DNA repair mechanisms. Methods: To investigate the role of complex SVs in HR-deficient cancers, we assembled a cohort of 2,367 WGS profiles from four tumor types (breast, ovarian, prostate, and pancreatic cancer) known to be associated with HR-deficiency. We identified 48 BRCA1-/- and 87 BRCA2-/- cases and called samples lacking (mono- or biallelic) mutations in BRCA1, BRCA2, or any other HR associated gene (e.g. PALB2, RAD51C) as wild-type. To validate observed structural variant patterns associated with homologous recombination deficiency from this larger dataset, a new cohort of 49 cases of invasive breast cancer with known BRCA1 (N = 29) or BRCA2 (N = 20) deficiency was collected as part of a prospective research study at Memorial Sloan Kettering Cancer Center and sequenced with 10X-linked read WGS in addition to standard Illumina short-read WGS. Results: Analysis of nearly 2,400 short-read whole genomes revealed distinct quasi-reciprocal structural variants (SVs) highly enriched in BRCA1-/- versus BRCA2-/- cancers. Applying high physical coverage (>150X) long molecule WGS to 49 tumor-normal pairs from breast cancer patients with inherited BRCA1 or BRCA2 loss-of-function mutations, we show that these SVs are associated with distinct cis or trans somatic allelic phases despite having nearly identical short read WGS footprints. Trans (crossover) outcomes were found to give rise to large-scale chromosomal variants that mediate the loss-of-heterozygosity patterns previously described as a cardinal feature of HR-deficient cancers. We find that these quasi-reciprocal SVs can be explained as distinct template switching outcomes from a shared intermediate arising after replication fork stalling in HR-deficient cancers. Furthermore, our WGS analyses reveal that BRCA2 genomes are highly enriched in deletions that harbor long (50-1000bp) tracts of inexact microhomology. These events, validated by long molecule WGS, indicate that single-stranded annealing serves as an active backup repair pathway in BRCA2-/- but not BRCA1-/- cancers. Conclusions: These results provide direct genomic evidence linking large-scale structural changes in HR-deficient tumors with specific backup repair pathways that suggest novel, therapeutically targetable dependencies. Our findings elucidate backup repair mechanisms responsible for generating structural variation in HR-deficient tumors, demonstrate the genotype-specific divergence of such compensatory DNA repair, and provide genomic features that improve the detection accuracy of HR-deficiency with utility for the optimal selection of treatment. Citation Format: Jeremy Setton, Kevin Hadi, Huasong Tian, Arnaud Da Cruz Paula, Joel Rosiene, Zi-Ning Choo, Julie Behr, Xiaotong Yao, Olivier Elemento, Britta Weigelt, Nadeem Riaz, Jorge S Reis-Filho, Marcin Imielinski, Simon N Powell. Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-04.

First report of Soybean Mosaic Virus in commercially grown soybean in the Netherlands.

In July 2020, plants with crinkled, chlorotic, occasionally necrotic leaves, typical for Soybean Mosaic Virus (SMV), were observed in eight soybean fields (Glycine max L.) in Flevoland, The Netherlands (Supp. Fig. 1). Disease incidence varied from 5-50% and the plants affected often occurred in small or extensive patches. Leaves from several symptomatic plants were sampled from each of two fields planted with soybean variety Green Shell or Summer Shell. Total RNA was extracted from one plant leaf sample per field using InviTrap Spin Plant RNA Mini Kit (Invitek, Germany). One-tube RT-PCRs employing potyvirus generic primers P9502 and CPUP (Van der Vlugt et al, 1999) and SMV-specific primers SMV-dT (5'-TTTTTTTTTTTTTTTAGGACAAC-3') and SMV-Nib-Fw (5'-CAAGGATGARTTTAAGGAG-3') combined with Sanger sequencing confirmed the presence of SMV in all leaf samples. To exclude the presence of other agents in the samples, total RNA from each cultivar was used in standard Illumina library preparation with ribosomal RNA depletion followed by sequencing on an Illumina NovaSeq6000 (paired-end, 150 bp) which yielded 66,579,158 reads (Summer Shell) and 223,953,206 reads (Green Shell). After quality trimming in CLC Genomics Workbench 20.0.4 (CLC-GWB; Qiagen, Hilden), four million reads were randomly sampled for de novo assembly. Contigs over 500 nucleotides (nts) in length with a minimum of 500 reads were annotated by BLASTn against NCBI GenBank. This identified one contig of 9,883 nts (6,233,397 reads) in Summer Shell and one contig of 9,727 nts (3,139,927 reads) in Green Shell with clear homology to SMV (E-value = 0.0). No other viruses were identified in the datasets. Reference assemblies against the SMV reference sequence (NC_002634) mapped 24,090,763 reads (36.2%) for Summer Shell and 175,459,637 reads (78.3%) for Green Shell. Extracted consensus sequences for SMV in both soybean cultivars were 9,584 nts long (excluding the poly-A tail). Sequence data from the de novo and reference assemblies were combined into consensus sequences which showed over 98% overall nt sequence identity to NC_002634 and 99.6% to each other. Both consensus sequences were deposited in GenBank under accession numbers MW822167 (SMV-Summer Shell) and MW822168 (SMV-Green Shell). In addition, the presence of SMV in the field samples was confirmed with an inoculation assay. Leaf samples from both fields were ground in phosphate buffer (0.1M, pH 7.2) and inoculated on cotyledons and first expanded leaves of soybean plants (unknown cv.) 12 days post-germination. Plants showed veinal chlorosis in systemic leaves from 12 days post-inoculation, which developed into veinal necrosis. SMV infections were confirmed by RT-PCR in systemic, chlorotic leaf samples of all symptomatic plants using the SMV-specific primers described above. To our knowledge, this is the first report of SMV in The Netherlands. As soybean is a relatively new but expanding crop in this country, information about emerging diseases is highly relevant. SMV can be transmitted via seeds and aphids, where seeds can serve as primary source of virus inoculum (Cui et al., 2011; Hartman et al., 2016; Hajimorad et al., 2018). Weeds and non-commercial plants can also serve as origin of SMV, particularly in subsequent growing seasons, although this virus infects a limited host range of six plant families (Cui et al., 2011; Hill & Whitham, 2014). Special monitoring would be advised for the recurrence and possible damage by SMV in Dutch soybean fields.

Genomic prediction in Nordic Red dairy cattle considering breed origin of alleles

This study investigated the reliability of genomic prediction (GP) using breed origin of alleles (BOA) approach in the Nordic Red (RDC) population, which has an admixed population structure. The RDC population consists of animals with varying degrees of genetic materials from the Danish Red (RDM), Swedish Red (SRB), Finnish Ayrshire (FAY), and Holstein (HOL) because bulls have been used across the breeds. The BOA approach was tested using 39,550 RDC animals in the reference population and 11,786 in the validation population. Deregressed proofs (DRP) of milk, fat and protein were used as response variable for GP. Direct genomic breeding values (DGV) for animals in the validation population were calculated with (BOA model) or without (joint model) considering breed origin of alleles. The joint model assumed homogeneous marker effects and a single set of marker effects were estimated, whereas BOA model assumed heterogeneous marker effects, and different sets of marker effects were estimated across the breeds. For the BOA approach, we tested scenarios assuming both correlated (BOA_cor) and uncorrelated (BOA_uncor) marker effects between the breeds. Additionally, we investigated GP using a standard Illumina 50K chip and including SNP selected from imputed whole-genome sequencing (50K+WGS). We also studied the effect of estimating (co)variances for genome regions of different sizes to exploit the information of the genome regions contributing to the (co)variance between the breeds. Region sizes were set as 1 SNP, a group of 30 or 100 adjacent SNP, or the whole genome. Reliability of DGV was measured as squared correlations between DGV and DRP divided by the reliability of DRP. Across the 3 traits, in general, RS30 and RS100 SNP yielded the highest reliabilities. Including WGS SNP improved reliabilities in almost all scenarios (0.297 on average for 50K and 0.307 on average for 50K+WGS). The BOA_uncor (0.233 on average) was inferior to the joint model (0.339 on average), but the reliabilities obtained using BOA_cor (0.334 on average) in most cases were not significantly different from those obtained using the joint model. The results indicate that both including additional whole-genome sequencing SNP and dividing the genome into fixed regions improve GP in the RDC. The BOA models have the potential to increase the reliability of GP, but the benefit is limited in populations with a high exchange of genetic material for a long time, as is the case for RDC.

Hackflex: low-cost, high-throughput, Illumina Nextera Flex library construction.

We developed a low-cost method for the production of Illumina-compatible sequencing libraries that allows up to 14 times more libraries for high-throughput Illumina sequencing to be generated for the same cost. We call this new method Hackflex. The quality of library preparation was tested by constructing libraries from Escherichia coli MG1655 genomic DNA using either Hackflex, standard Nextera Flex (recently renamed as Illumina DNA Prep) or a variation of standard Nextera Flex in which the bead-linked transposase is diluted prior to use. In order to test the library quality for genomes with a higher and a lower G+C content, library construction methods were also tested on Pseudomonas aeruginosa PAO1 and Staphylococcus aureus ATCC 25923, respectively. We demonstrated that Hackflex can produce high-quality libraries and yields a highly uniform coverage, equivalent to the standard Nextera Flex kit. We show that strongly size-selected libraries produce sufficient yield and complexity to support de novo microbial genome assembly, and that assemblies of the large-insert libraries can be much more contiguous than standard libraries without strong size selection. We introduce a new set of sample barcodes that are distinct from standard Illumina barcodes, enabling Hackflex samples to be multiplexed with samples barcoded using standard Illumina kits. Using Hackflex, we were able to achieve a per-sample reagent cost for library prep of A$7.22 (Australian dollars) (US $5.60; UK £3.87, £1=A$1.87), which is 9.87 times lower than the standard Nextera Flex protocol at advertised retail price. An additional simple modification and further simplification of the protocol by omitting the wash step enables a further price reduction to reach an overall 14-fold cost saving. This method will allow researchers to construct more libraries within a given budget, thereby yielding more data and facilitating research programmes where sequencing large numbers of libraries is beneficial.

Next Generation Sequencing of DNA-Encoded Libraries.

DEL technology is dependent on the generation and analysis of large amounts of DNA sequence data. In this chapter, we describe a method to sequence DEL libraries that uses a customized preparative PCR protocol, along with standard steps for purification, analysis, and sequencing of the amplified library DNA on an Illumina sequencing platform. Compared with standard Illumina sequencing library preparation protocols, in which a PCR reaction is followed by end repair, adenylation, ligation of Illumina adapters, and a second preparative PCR reaction, the customized operations described here provide significantly improved process efficiency and sequencing quality.

Development of a User-Friendly Pipeline for Mutational Analyses of HIV Using Ultra-Accurate Maximum-Depth Sequencing.

Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.

Viral metagenomics reveals the presence of novel Zika virus variants in Aedes mosquitoes from Barbados

BackgroundThe Zika virus (ZIKV) epidemic of 2015/2016 spread throughout numerous countries. It emerged in mainland Latin America and spread to neighboring islands, including the Caribbean island of Barbados. Recent studies have indicated that the virus must have already been circulating in local mosquito populations in Brazil for almost 2 years before it was identified by the World Health Organization in 2015. Metagenomic detection assays have the potential to detect emerging pathogens without prior knowledge of their genomic nucleic acid sequence. Yet their applicability as vector surveillance tools has been widely limited by the complexity of DNA populations from field-collected mosquito preparations. The aim of this study was to investigate local vector biology and characterize metagenomic arbovirus diversity in Aedes mosquitoes during the ongoing 2015/2016 ZIKV epidemic.MethodsWe performed a short-term vector screening study on the island of Barbados during the ongoing 2015/2016 ZIKV epidemic, where we sampled local Aedes mosquitoes. We reanalyzed mosquito viral microbiome data derived from standard Illumina MiSeq sequencing to detect arbovirus sequences. Additionally, we employed deep sequencing techniques (Illumina HiSeq) and designed a novel bait capture enrichment assay to increase sequencing efficiency for arbovirus sequences from complex DNA samples.ResultsWe found that Aedes aegypti seemed to be the most likely vector of ZIKV, although it prevailed at a low density during the observed time period. The number of detected viruses increased with sequencing depth. Arbovirus sequence enrichment of metagenomic DNA preparations allowed the detection of arbovirus sequences of two different ZIKV genotypes, including a novel one. To our knowledge, this is the first report of the S3116W mutation in the NS5 gene region of ZIKV polyprotein.ConclusionsThe metagenomic arbovirus detection approach presented here may serve as a useful tool for the identification of epidemic-causing arboviruses with the additional benefit of enabling the collection of phylogenetic information on the source. Apart from detecting more than 88 viruses using this approach, we also found evidence of novel ZIKV variants circulating in the local mosquito population during the observed time period.Graphical abstract

Genomic and Outcome Analyses of Philadelphia Chromosome-like (Ph-like) NCI Standard Risk B-Acute Lymphoblastic Leukemia (SR B-ALL) Patients Treated on Children's Oncology Group (COG) AALL0331

Introduction: Ph-like ALL is defined by a gene expression profile similar to Philadelphia chromosome-positive ALL but lacking the BCR - ABL1 fusion. Ph-like ALL is comprised of diverse genomic alterations activating kinase and cytokine receptor signaling, including chimeric fusions involving ABL1 and related genes (ABL2, CSF1R, PDGFRB ; ABL-class fusions), genomic rearrangements leading to CRLF2 over-expression, JAK2 gene fusions, and truncating rearrangements of EPOR . We have published several large studies describing the clinical features, treatment outcome, and genomic alterations in Ph-like ALL that focused on patients with NCI high risk (HR) ALL (age ≥10 years or WBC ≥50,000) or those with SR ALL (age 1-9.99 years and WBC <50,000) and elevated minimal residual disease (MRD) at the end of induction (EOI; Reshmi, BLOOD 2017 and Roberts, NEJM 2014). The purpose of the current study was to assess the frequency of genomic lesions in Ph-like SR ALL patients lacking HR features.Methods: We retrospectively analyzed 1023 unselected newly-diagnosed SR ALL patients enrolled on COG AALL0331 between 2006 and 2008, and compared characteristics of SR Ph-like cases to the HR patients described in our prior studies. All cases were screened using an 8-gene Taqman low-density array (LDA) PCR assay to identify the Ph-like GEP (Harvey, ASH 2013). Ph-like cases with elevated CRLF2 expression were tested for CRLF2 rearrangement (CRLF2 -R; P2RY8 - CRLF2 by Taqman PCR on the LDA card and IGH - CRLF2 by FISH). JAK mutations in CRLF2 -R cases were tested by Sanger sequencing. Ph-like cases without CRLF2 -R were tested for previously identified kinase fusions involving ABL1, ABL2, CSF1R, JAK2, NTRK3, and PDGFRB by RT-PCR. A subset of Ph-like cases without detected genomic alterations underwent RNA-sequencing using standard Illumina library preparation.Results: A total of 205 patients had LDA value ≥0.5, with 145 (14.2%) remaining after exclusion of BCR-ABL1 and ETV6-RUNX1 . Of these 145, 85 were CRLF2high (57.9%) as defined by CRLF2 expression levels on the LDA card. Among these 85, 42 (50%) had CRLF2 -R, compared to 80% in HR patients. P2RY8 - CRLF2 was seen in 36 cases, IGH - CRLF2 in 4 and 2 had CRLF2 -R with an unknown partner; 14 (32.6%) of these had a concomitant JAK mutation. Of the 60 Ph-like CRLF2low cases, five had a targetable kinase fusion identified by RT-PCR or RNA sequencing: two ABL - class fusions (ZMIZ1 - ABL1, EBF1-PDGFRB); one PAX5-JAK2 fusion; one ETV6-JAK2 fusion; and one ETV6 - NTRK3 fusion. The 145 Ph-like cases had inferior 7-year event-free survival (EFS; 83.1±3.5% vs. 90.6 ±0.1%; P=0.005) and non-significantly inferior 7-year overall survival (OS; 93.5±2.3% vs. 95.5 ±0.8%; P=0.184) compared to 869 non Ph-like cases. Ph-like ALL cases were more likely to have end induction MRD ≥ 0.01% (24.3% vs. 17.9%; P=0.049), and positive MRD was predictive of outcome for Ph-like (7-year EFS: 73.2±8.3%, vs. 86.6 ±3.7%; P=0.079; 7-year OS: 88.2 ±5.9% vs. 95.9±2.1%; P=0.162) and non Ph-like patients (7-year EFS: 79.7 ±3.7% vs. 93.3 ±1.0%; P <0.0001; 7-year OS: 90.2% SE 2.7 vs. 97.4±0.9%; P <0.0001). In multivariate analyses, Ph-like and MRD status EOI were significant predictors of EFS (p=0.016; <0.0001 respectively), while only MRD had a significant impact on OS.Conclusion: Ph-like ALL is less common in NCI SR than HR patients with significant differences in the frequency of various genomic alterations in Ph-like ALL cases based on NCI risk group. Our previous studies have shown that Ph-like ALL HR patients have a substantially increased risk of treatment failure and death as compared to non Ph-like HR patients (Mullighan, NEJM 2009 and Roberts, NEJM 2014). This cohort of SR Ph-like patients had a significantly inferior EFS and a trend to inferior OS, but the magnitude of effect was much smaller than that seen in HR Ph-like patients, with overall outcomes much better in SR as compared to HR Ph-like patients. These findings underscore the significant differences in Ph-like ALL frequency, genetic subtypes, and outcome based on age and initial WBC. [Display omitted] DisclosuresBorowitz:Beckman Coulter: Honoraria; Becton-Dickinson Biosciences: Research Funding; HTG Molecular: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Mattano:HARP Pharma Consulting, LLC: Employment. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Hunger:Amgen: Consultancy, Equity Ownership; Novartis: Consultancy; Erytech Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Honoraria.

Ultra-accurate microbial amplicon sequencing with synthetic long reads

BackgroundOut of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge.MethodsHere, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads.ResultsLoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens.ConclusionsThe order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.4PHa9SHy5rS1yXExpPUL7_Video abstract.

10 STARTUPS TO WATCH 2021: Early-stage companies advance precision medicine by applying AI, clinical genomics, and other new technologies

10 STARTUPS TO WATCH 2021: Early-stage companies advance precision medicine by applying AI, clinical genomics, and other new technologies

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

BackgroundK-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design.ResultsAs the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%.ConclusionK-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

ReSeq simulates realistic Illumina high-throughput sequencing data

In high-throughput sequencing data, performance comparisons between computational tools are essential for making informed decisions at each step of a project. Simulations are a critical part of method comparisons, but for standard Illumina sequencing of genomic DNA, they are often oversimplified, which leads to optimistic results for most tools. ReSeq improves the authenticity of synthetic data by extracting and reproducing key components from real data. Major advancements are the inclusion of systematic errors, a fragment-based coverage model and sampling-matrix estimates based on two-dimensional margins. These improvements lead to more faithful performance evaluations. ReSeq is available at https://github.com/schmeing/ReSeq.

Rapid and Detailed Characterization of Transgene Insertion Sites in Genetically Modified Plants via Nanopore Sequencing.

Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species—perennial ryegrass, white clover, and canola—were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective.

Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms

BackgroundThe increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated.ResultsHere, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries.ConclusionsOur approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Improved genomic resources for the black tiger prawn (Penaeus monodon)

World production of farmed crustaceans was 7.8 million tons in 2016. While only making up approximately 10% of world aquaculture production, crustaceans are generally high-value species and can earn significant export income for producing countries. Viet Nam is a major seafood producing country earning USD 7.3 billion in 2016 in export income with shrimp as a major commodity. However, there is a general lack of genomic resources available for shrimp species, which is challenging to obtain due to the need to deal with large repetitive genomes, which characterize many decapod crustaceans. The first tiger prawn (P. monodon) genome assembly was assembled in 2016 using the standard Illumina PCR-based pair-end reads and a computationally-efficient but relatively suboptimal assembler, SOAPdenovo v2. As a result, the current P. monodon draft genome is highly fragmented (> 2 million scaffolds with N50 length of <1000 bp), exhibiting only moderate genome completeness (< 35% BUSCO complete single-copy genes). We sought to improve upon the recently published P. monodon genome assembly and completeness by generating Illumina PCR-free pair-end sequencing reads to eliminate genomic gaps associated with PCR-bias and performing de novo assembly using the updated MaSurCA de novo assembler. Furthermore, we scaffolded the assembly with low coverage Nanopore long reads and several recently published deep Illumina transcriptome paired-end sequencing data, producing a final genome assembly of 1.6 Gbp (1,211,364 scaffolds; N50 length of 1982 bp) with an Arthropod BUSCO completeness of 96.8%. Compared to the previously published P. monodon genome assembly from China (NCBI Accession Code: NIUS01), this represents an almost 20% increase in the overall BUSCO genome completeness that now consists of more than 90% of Arthropod BUSCO single-copy genes. The revised P. monodon genome assembly (NCBI Accession Code: VIGR01) will be a valuable resource to support ongoing functional genomics and molecular-based breeding studies in Vietnam.

UNAGI: an automated pipeline for nanopore full-length cDNA sequencing uncovers novel transcripts and isoforms in yeast

Sequencing the entire RNA molecule leads to a better understanding of the transcriptome architecture. SMARTer (Switching Mechanism at 5′-End of RNA Template) is a technology aimed at generating full-length cDNA from low amounts of mRNA for sequencing by short-read sequencers such as those from Illumina. However, short read sequencing such as Illumina technology includes fragmentation that results in bias and information loss. Here, we built a pipeline, UNAGI or UNAnnotated Gene Identifier, to process long reads obtained with nanopore sequencing and compared this pipeline with the standard Illumina pipeline by studying the Saccharomyces cerevisiae transcriptome in full-length cDNA samples generated from two different biological samples: haploid and diploid cells. Additionally, we processed the long reads with another long read tool, FLAIR. Our strand-aware method revealed significant differential gene expression that was masked in Illumina data by antisense transcripts. Our pipeline, UNAGI, outperformed the Illumina pipeline and FLAIR in transcript reconstruction (sensitivity and specificity of 80% and 40% vs. 18% and 34% and 79% and 32%, respectively). Moreover, UNAGI discovered 3877 unannotated transcripts including 1282 intergenic transcripts while the Illumina pipeline discovered only 238 unannotated transcripts. For isoforms profiling, UNAGI also outperformed the Illumina pipeline and FLAIR in terms of sensitivity (91% vs. 82% and 63%, respectively). But the low accuracy of nanopore sequencing led to a closer gap in terms of specificity with Illumina pipeline (70% vs. 63%) and to a huge gap with FLAIR (70% vs 0.02%).