Illumina Systems Research Articles

IS1-related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli.

Nitrofurantoin is a broad-spectrum first-line antimicrobial used for managing uncomplicated urinary tract infection (UTI). Loss-of-function mutations in chromosomal genes nfsA, nfsB and ribE of Escherichia coli are known to reduce nitrofurantoin susceptibility. Here, we report the discovery of nitrofurantoin heteroresistance in E. coli clinical isolates and a novel genetic mechanism associated with this phenomenon. Subpopulations with lower nitrofurantoin susceptibility than major populations (hereafter, nitrofurantoin-resistant subpopulations) in two E. coli blood isolates (previously whole-genome sequenced) were identified using population analysis profiling. Each isolate was known to have a loss-of-function mutation in nfsA. From each isolate, four nitrofurantoin-resistant isolates were derived at a nitrofurantoin concentration of 32 mg l-1, and a comparator isolate was obtained without any nitrofurantoin exposure. Genomes of derived isolates were sequenced on Illumina and Nanopore MinION systems. Genetic variation between isolates was determined based on genome assemblies and read mapping. Nitrofurantoin minimum inhibitory concentrations (MICs) of both blood isolates were 64 mg l-1, with MICs of major nitrofurantoin-susceptible populations varying from 4 to 8 mg l-1. Two to 99 c.f.u. per million demonstrated growth at the nitrofurantoin concentration of 32 mg l-1, which is distinct from that of a homogeneously susceptible or resistant isolate. Derived nitrofurantoin-resistant isolates had 11-66 kb deletions in chromosomal regions harbouring nfsB, and all deletions were immediately adjacent to IS1-family insertion sequences. Our findings demonstrate that the IS1-associated large-scale genetic deletion is a hitherto unrecognized mechanism of nitrofurantoin heteroresistance and could compromise UTI management. Further, frequencies of resistant subpopulations from nitrofurantoin-heteroresistant isolates may challenge conventional nitrofurantoin susceptibility testing in clinical settings.

1798. Phenotypic and Genotypic Impact of Antibiotic Stewardship Intervention on Daptomycin-Nonsusceptible Enterococcus faecium (DNSE) Clinical Isolates

BackgroundSince its introduction in 2009, use of daptomycin for treatment of enterococcal infections has resulted in the emergence of DNSE. Between 2009 and 2013, daptomycin nonsusceptibility among E. faecium was closely associated with emergence of a unique and dominant clone ST736 in our institution. In 2014, we instituted targeted measures to optimize the use of daptomycin. In this study, we describe the significant phenotypic and genotypic impact of reduced daptomycin use on clinical enterococcal isolates.MethodsEnterococcal clinical isolates were recovered from January 2014 through December 2017. Daptomycin susceptibility was determined by MicroScan WalkAway™ System and confirmed by E-test. Selected DNSE and vancomycin-resistant E. faecium (VREfm) clinical isolates were analyzed by next-generation sequencing (NGS) using the Illumina systems to provide multilocus sequencing type (MLST). Daptomycin utilization data were extracted from pharmacy records.ResultsTargeted antibiotic stewardship initiatives consisted of preapproval, daily review for optimization of dose and duration, rapid de-escalation, consideration for appropriate alternative antibiotics for select disease syndromes and stopping of inappropriate daptomycin therapy. Over 4 years, this lead to a 39% reduction in overall use of daptomycin. Besides direct cost saving, this reduced use was associated with significant reduction in daptomycin nonsusceptibility from 12% to 4%, lowering of MIC90 from 8 to 4 μg/mL, and a clonal shift from dominant ST736 to ST117.Daptomycin Usage Days of Therapy /1,000 Days (Monthly Average)Phenotypic ChangesGenotypic ChangesMIC50 (μg/mL)MIC90 (μg/mL)% DNSEDominant Genotype Among VREfm20147424812 ST736 (38%)ST18 (21%)ST412 (12%)ST117 (11%)2015633461020165764472017455444ST736 (5%)ST18 (4%)ST412 (11%)ST117 (41%)ConclusionA targeted antibiotic stewardship initiative to address rising rate of daptomycin nonsusceptibility among E. faecium, resulted in significant phenotypic and genotypic changes among clinical isolates. This study also shows successful integration of NGS in a clinical microbiology lab to validate phenotypic changes of daptomycin nonsusceptibility and to help design future infection control and antibiotic stewardship endeavors.Disclosures A. Dhand, Merck: Speaker’s Bureau, Speaker honorarium. Astellas: Scientific Advisor, Consulting fee.

Abstract 1847: Evaluating the metastatic potential and the molecular heterogeneity of patient-derived orthotopic xenograft models of triple-negative breast cancer

Abstract We report an in-depth characterization of patient-derived orthotopic xenograft (PDOX) models of triple-negative breast cancer (TNBC) regarding their molecular profile at the single cell level, tumor heterogeneity, 3D organoid generation, and ability to generate circulating tumor cells (CTCs). A panel of seven TNBC PDOX tumors were grown orthotopically in Nod scid gamma mice and used in this study. Blood obtained via cardiac puncture from tumor bearing animals was processed on the Vortex microfluidic platform, for label-free, size-based enrichment of circulating tumor cells (CTCs). Enriched cell populations were stained for human-specific cytokeratin (CK) and Vimentin (Vim), mouse-specific CD45, and DAPI; CTCs were identified as cells that were CD45 negative and positive for CK or Vim. Bulk tumor growing in the mammary fat pads was dissociated to single cells and characterized using Fluidigm’s® PolarisTM platform for single cell biological experimentation and cDNA generation within an integrated fluidic circuit (IFC). From the cell suspension, Polaris identified single cells that were then processed for mRNA-seq. The resulting cDNA libraries were then multiplexed using Nextera XT® (Illumina®) and sequenced on Illumina systems. Data generated from mRNA-seq was processed to correct for confounding factors such as cell size, cell cycle and read depth and then analyzed to screen for heterogeneity between different populations of cells. Tumors were analyzed by flow cytometry for both tumor and immune cells and additionally the single cell suspension was seeded into 3-D culture to generate organoids. Finally, organs from tumor bearing animals were analyzed for metastases. With the Vortex platform, we detected CTCs from a majority of our PDOX tumor-bearing mice. The total number of CTCs varied over a wide range between different PDOX tumors. There was a clear heterogeneity in CTCs in terms of CK and Vim expression. In CTCs from one of the PDOX tumors, we detected a small population of CTCs that were either CK+ or Vim+ but the major fraction that was double positive (Vim+ CK+). Probing the bulk tumor from different PDOX models revealed heterogeneity in the levels and number of cells positive for cell surface markers like EpCAM and a difference in the levels of infiltrating myeloid cells (CD11b+). mRNA-seq analyses of individual tumor cells from the bulk tumor belonging to different PDOX models will be described. Additionally, lung and brain metastases were identified. 3D organoid cultures from our PDOX models were successfully grown and their gene expression profiles will be analyzed. In summary, PDOX models of TNBC will help advance our understanding of the molecular basis of this deadly cancer. Citation Format: Vishnu C. Ramani, Rakhi Gupta, Gerald Quon, Melanie Triboulet, Corinne Renier, Cassandra Greene, Chad Sanada, Tracy Lu, Lukasz Szpankowski, Naveen Ramalingam, Ameen A. Salahudeen, Sean de la O, Ranjani Rajapaksa, Shoshana Levy, Anne A. Leyrat, Jay A. West, Elodie Sollier-Christen, Calvin J. Kuo, George W. Sledge, Stefanie S. Jeffrey. Evaluating the metastatic potential and the molecular heterogeneity of patient-derived orthotopic xenograft models of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1847. doi:10.1158/1538-7445.AM2017-1847

Sequencing on the SOLiD 5500xl System – in-depth characterization of the GC bias

ABSTRACT Different types of sequencing biases have been described and subsequently improved for a variety of sequencing systems, mostly focusing on the widely used Illumina systems. Similar studies are missing for the SOLiD 5500xl system, a sequencer which produced many data sets available to researchers today. Describing and understanding the bias is important to accurately interpret and integrate these published data in various ongoing research projects. We report a particularly strong GC bias for this sequencing system when analyzing a defined gDNA mix of 5 microbes with a wide range of different GC contents (20–72%) when comparing to the expected distribution and Illumina MiSeq data from the same DNA pool. Since we observed this bias already under PCR-free conditions, changing the PCR conditions during library preparation – a common strategy to handle bias in the Illumina system - was not relevant. Source of the bias appeared to be an uneven heat distribution during the SOLiD emulsion PCR (ePCR) - for enrichment of libraries prior loading – since ePCR in either small pouches or in 96-well plates improved the GC bias. Sequencing of chromatin immunoprecipitated DNA (ChIP-seq) is a common approach in epigenetics. ChIP-seq of the mixed source histone mark H3K9ac (acetyl Histone H3 lysine 9), typically found on promoter regions and on gene bodies, including CpG islands, performed on a SOLiD 5500xl machine, resulted in major loss of reads at GC rich loci (GC content ≥ 62%), not explained by low sequencing depth. This was improved with adaptations of the ePCR.

Flexbar 3.0 - SIMD and multicore parallelization.

High-throughput sequencing machines can process many samples in a single run. For Illumina systems, sequencing reads are barcoded with an additional DNA tag that is contained in the respective sequencing adapters. The recognition of barcode and adapter sequences is hence commonly needed for the analysis of next-generation sequencing data. Flexbar performs demultiplexing based on barcodes and adapter trimming for such data. The massive amounts of data generated on modern sequencing machines demand that this preprocessing is done as efficiently as possible. We present Flexbar 3.0, the successor of the popular program Flexbar. It employs now twofold parallelism: multi-threading and additionally SIMD vectorization. Both types of parallelism are used to speed-up the computation of pair-wise sequence alignments, which are used for the detection of barcodes and adapters. Furthermore, new features were included to cover a wide range of applications. We evaluated the performance of Flexbar based on a simulated sequencing dataset. Our program outcompetes other tools in terms of speed and is among the best tools in the presented quality benchmark. https://github.com/seqan/flexbar. johannes.roehr@fu-berlin.de or knut.reinert@fu-berlin.de.

An adaptive decorrelation method removes Illumina DNA base-calling errors caused by crosstalk between adjacent clusters

Base-calling accuracy is crucial for high-throughput DNA sequencing and downstream analysis such as read mapping and genome assembly. Accordingly, we made an endeavor to reduce DNA sequencing errors of Illumina systems by correcting three kinds of crosstalk in the cluster intensity data. We discovered that signal crosstalk between adjacent clusters accounts for a large portion of sequencing errors in Illumina systems, even after correcting color crosstalk caused by the overlap of dye emission spectra and phasing/pre-phasing caused by out-of-step nucleotide synthesis. Interestingly and importantly, spatial crosstalk between adjacent clusters is cluster-specific and often asymmetric, which cannot be corrected by existing deconvolution methods. Therefore, we introduce a novel mathematical method able to estimate and remove spatial crosstalk, thereby reducing base-calling errors by 44–69% at a given mapping rate from Illumina systems. Furthermore, the resolution gained from this work provides new room for higher throughput of DNA sequencing and of general measurement systems using fluorescence-based imaging technology. The resulting base-caller 3Dec is available for academic users at http://github.com/flishwnag/3dec. Not only does it reduce 62.1% errors compared to the standard pipeline, but also its implementation is fast enough for daily sequencing.

Abstract A14: The transcriptomic landscape of the two emblematic colorectal cancer cell lines HCT116 and HT29

Abstract Colorectal cancer (CRC) is the third leading cause of malignant mortality worldwide, accounting for more than 13% of all cancer death. CRC harbors several alterations, mainly, two major classes of these instability were described: 1/microsatellite instability (MSI) caused by the inactivation and loss of expression of the mismatch repair genes (MMR), and 2/chromosome instability or microsatellite stable (MSS). CRC Cell lines are invaluable biomedical research tools to study these alterations. Two emblematic cell lines for CRC were extensively used, HT29 for MSS and HCT116 for MSI. Short and long reads, high coverage whole transcriptome sequencing of these two cell lines and two normal cell lines, enable us to establish the signaling pathways altered in these two cancer cell lines. Method: Total RNA were extracted from four cell lines HT29, HCT116, CR1780 and CRL18 using RNeasy Mini Kit (Qiagen). mRNA libraries were generated using TruSeq Stranded mRNA Library Prep Kit (Illumina), and high-throughput sequencing were performed using Illumina Systems. Pair-end reads of 100bp and 300bp were generated by Hiseq and Miseq, respectively. All reads were filtered for high-quality reads (≥Q30, adapters trimming and broken pair-reads removing). Reads alignment and genes profiling analysis were done with CLCbio software. Genes upregulated and downregulated with p-value <0.05, in cancer cell lines versus normal cell lines and between the two cancer cell lines were analyzed by Ingenuity Pathways Analysis (IPA). Results: The transcriptome analysis reveals 11,564 different splice variants are differentially expressed in both cancer cell lines HT29 and HCT116, when compared to the two normal cell lines (p-value <0.05). Among which, 9041 transcripts are upregulated and 2523 downregulated. The comparison between HT29 and HCT116 reveals only 333 splice variants differentially expressed. The canonical signaling pathways analysis demonstrates extensive molecular heterogeneity in cancer cell lines. in which, two major pathways clearly appear, mTOR signaling pathway (mTORsp) and estrogen receptor signaling pathway (ERsp). Half or more the number of molecules, which constitute these two pathways, has altered transcription expression, 96 genes among 194 molecules of mTORsp, and 66 genes among 127 molecules of ERsp. Interestingly, when comparing cancer cell lines with each other, 47 genes among 194 of mTORsp have altered expression and no gene from ERsp was found. Conclusion: Our finding established the signaling pathways altered in HT29 and HCT116 and shows the importance of mTOR and estrogen receptor signaling pathways in these two cancer cell lines. Citation Format: Hicham Mansour, Noureddine BOUAROUROU, Jean-Pierre Roperch, Faouzi Habib, Mourad Said. The transcriptomic landscape of the two emblematic colorectal cancer cell lines HCT116 and HT29. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr A14.

Abstract B08: Potential biomarkers for colorectal cancer diagnosis and prognosis

Abstract Colorectal cancer (CRC) is one of the most common cancers worldwide. It harbors several genetic and epigenetic alterations, which makes finding suitable biomarkers a challenge. CRC Cell lines are invaluable biomedical research tools for drug screening and biomarker discovery. Two emblematic cell lines for CRC were extensively used, HT29 and HCT116. We performed the whole transcriptome sequencing of these two cell lines and two normal epithelial cell lines, CR1780 and CRL18, derived from the colon as control. A methylation quantification of downregulated genes enables to screen potential biomarkers, which could be used as potential biomarkers for CRC diagnosis and prognosis. Method: Total RNA was extracted from HT29, HCT116, CR1780 and CRL18 using RNeasy Mini Kit (Qiagen). mRNA libraries were generated using TruSeq Stranded mRNA Library Prep Kit (Illumina), and high-throughput sequencing were performed using Illumina systems (Hiseq and Miseq). All reads were filtered for high-quality reads (≥Q30, adapters trimming and broken pair-reads removing). Reads alignment and genes profiling analysis were done with CLCbio software. Selected genes downregulated with p-value <0.001 in cancer cell lines versus normal cell lines, were evaluated for methylation in DNA extracted from tumors and autologous normal tissues from CRC patients. Results: The transcriptome analysis reveals 741 different splice variants are downregulated in both cancer cell lines HT29 and HCT116, when compared to the two normal cell lines, CR1780 and CRL18 (p-value <0.001). 10 relevant downregulated genes were selected, ACTG2, THY1, IGFBP3, WNT2, RAB32, SPARC, COL1A, MMP2, IGFBP3 and VIM. Interestingly, all the splice variants (10) of Vim, 7 of ATG2, 4 of THY1 and 2 of IGFBP3 were dramatically downregulated. Methylation quantification demonstrates the hypermethylation of all these 10 downregulated genes, in the tumors compared to matched normal tissues. Conclusion. At the level of mRNA expression and methylation, this study enables to screen potential biomarkers for CRC diagnosis and prognosis. Citation Format: Hicham Mansour, Noureddine Bouarourou, Jean Pierre Roperch, Fouazi Habib, Mourad Said. Potential biomarkers for colorectal cancer diagnosis and prognosis. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B08.

Achieving Accurate and Fast Base-calling by a Block Model of the Illumina Sequencing Data

Base-calling accuracy is crucial for high-throughput DNA sequencing and we considered the de-convolution of the intensity data of Illumina systems. While the phasing and color crosstalk are considered with fixed parameters in existing base-calling softwares, we found that these processes are not constant in different part of the data. We thus model and deal with the data in blocks, and by this approach we improve the base calling accuracy as well as reduce the model complexity. A block method based on a linear statistical model is then developed, which balances the accuracy against the computational speed. The performances under different blocking conditions are also analyzed.

Abstract 3564: A new method for preparation of low-input, PCR-free next generation sequencing libraries

Abstract Sequence analysis of PCR-free Next Generation Sequencing (NGS) libraries demonstrates improved evenness of coverage relative to NGS libraries that have undergone PCR amplification. However, existing PCR-free methods require 1ug or more of input DNA quantity in order to obtain sufficient library yield which restricts such analysis when DNA sample quantity is insufficient. To expand the applicability of PCR-free sequencing, Swift Biosciences has developed a novel adapter attachment chemistry that is more efficient than existing methods. This new technology, Accel-NGS™, results in libraries that capture a higher complexity of each sample which enables a lower input DNA requirement. For Illumina systems, PCR-free Accel-NGS libraries can be prepared from 100ng of input DNA for direct sequencing. PCR-free Accel-NGS libraries demonstrate even coverage across a broad GC content as well as when analyzing whole genome sequence coverage. To enable library preparation from even less DNA, Accel-NGS libraries can also be prepared with a low number of PCR cycles for direct sequencing. The Accel-NGS method with PCR results in more even coverage and less PCR duplicates when compared to existing methods that use PCR. For exome hybridization capture, comparable coverage was achieved for Accel-NGS libraries from 100ng DNA input when compared to an existing method that requires 1ug of DNA input. The Accel-NGS method is fast and automatable; additional advantages when applied to clinical samples are under investigation. PCR-free Accel-NGS libraries can also be prepared for Ion Torrent systems from 5ng of input DNA with comparable sequence quality to existing methods. Citation Format: Laurie Kurihara, L. Banks, S. Chupreta, C. Couture, V. Kelchner, J.Laliberte, S. Sandhu, R. Spurbeck, V. Makarov. A new method for preparation of low-input, PCR-free next generation sequencing libraries. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2014-3564

Genome sequence of type strain of Staphylococcus aureus subsp. aureus

BackgroundStaphylococcus aureus is a pathogen that causes food poisoning and community-associated infection with antibiotic resistance. This species is an indigenous intestinal microbe found in infants and not found in adult intestine. The relatively small genome size and rapid evolution of antibiotic resistance genes in the species have been drawing an increasing attention in public health. To extend our understanding of the species and use the genome data for comparative genomic studies, we sequenced the type strain of S. aureus subsp. aureus DSM 20231T.ResultsSeventeen contigs were generated using hybrid assembly of sequences derived from the Roche 454 and Illumina systems. The length of the genome sequence was 2,902,619 bases with a G + C content of 32.8%. Among the 2,550 annotated CDSs, 44 CDSs were annotated to antibiotic resistance genes and 13 CDSs were related to methicillin resistance. It is interesting to note that this strain was first isolated in 1884 before methicillin was generally used on patients.ConclusionsThe present study analyzed the genome sequence of S. aureus subsp. aureus type strain as the reference sequence for comparative genomic analyses of clinical isolates. Methicillin resistance genes found in the genome indicate the presence of antibiotic resistance mechanism prior to the usage of antibiotics. Further comparative genomic studies of methicillin-resistant strains based on this reference genome would help to understand the evolution of resistance mechanism and dissemination of resistance genes.

Evaluating methods for ranking differentially expressed genes applied to microArray quality control data

BackgroundStatistical methods for ranking differentially expressed genes (DEGs) from gene expression data should be evaluated with regard to high sensitivity, specificity, and reproducibility. In our previous studies, we evaluated eight gene ranking methods applied to only Affymetrix GeneChip data. A more general evaluation that also includes other microarray platforms, such as the Agilent or Illumina systems, is desirable for determining which methods are suitable for each platform and which method has better inter-platform reproducibility.ResultsWe compared the eight gene ranking methods using the MicroArray Quality Control (MAQC) datasets produced by five manufacturers: Affymetrix, Applied Biosystems, Agilent, GE Healthcare, and Illumina. The area under the curve (AUC) was used as a measure for both sensitivity and specificity. Although the highest AUC values can vary with the definition of "true" DEGs, the best methods were, in most cases, either the weighted average difference (WAD), rank products (RP), or intensity-based moderated t statistic (ibmT). The percentages of overlapping genes (POGs) across different test sites were mainly evaluated as a measure for both intra- and inter-platform reproducibility. The POG values for WAD were the highest overall, irrespective of the choice of microarray platform. The high intra- and inter-platform reproducibility of WAD was also observed at a higher biological function level.ConclusionThese results for the five microarray platforms were consistent with our previous ones based on 36 real experimental datasets measured using the Affymetrix platform. Thus, recommendations made using the MAQC benchmark data might be universally applicable.

Abstract 1164: Parallel preparation of targeted resequencing libraries from 480 genomic regions using multiplex PCR on the Access Array system

Abstract Next generation sequencing platforms have dramatically reduced sequencing costs. However, it currently remains too expensive to routinely resequence entire human genomes in order to discover genetic variants or somatic mutations underlying tumorigenesis. Therefore, a need exists for multiplexed, targeted amplification methods that allow for the analysis of multiple genomic regions in large cohorts. Available targeted enrichment technologies are either aimed at the capture of regions of interest from a single sample, exhibit uneven representation or require significant amounts of input material. The novel microfluidic platform, the Access Array system, combines 48 samples with 48 primer sets resulting in 2,304 simultaneously occurring PCR amplifications requiring as little as 50ng DNA per sample. PCR products generated on the Access Array system can be used for sequencing on all next-generation sequencing platforms, including 454 GS FLX and Illumina GAII. To increase coverage and throughput, PCR reactions can be multiplexed within Access Array chips generating up to 480 amplicons per sample. As proof-of-principal experiments, we have carried out multiplexed amplifications of a set of commonly mutated cancer gene exons across 48 genomic DNA samples. In initial experiments, 580 primer pairs were validated in individual PCR reactions in 96-well plates. Each primer pair was designed to include 5’ sequences that allow for the incorporation of 454 and Illumina adapters necessary for subsequent emPCR and cluster generation, respectively. 480 primer pairs that produced a single band of the correct size, as determined on a Caliper LabChip system, were selected for multiplex PCR experiments. Primer pairs yielding amplicons with a similar size were combined in groups of 10 sets, resulting in 48 primer pools of 10 primer pairs each. Multiplex PCR was carried out on Access Array chips, followed by harvesting of the 48 amplicon pools. Each pool was diluted and then subjected to a second round of PCR in standard 96-well plates with barcoded universal primers corresponding to the 454 and Illumina sequences. The resulting products are 48 uniquely barcoded amplicon pools, each comprising 480 amplicons derived from one sample, that are ready for sequencing. We will present sequencing data generated on both 454 and Illumina systems. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1164.