Meredith Ashby

A hybrid approach for the automated finishing of bacterial genomes

Advances in DNA sequencing technology have improved our ability to characterize most genomic diversity. However, accurate resolution of large structural events is challenging because of the short read lengths of second-generation technologies. Third-generation sequencing technologies, which can yield longer multikilobase reads, have the potential to address limitations associated with genome assembly. Here we combine sequencing data from second- and third-generation DNA sequencing technologies to assemble the two-chromosome genome of a recent Haitian cholera outbreak strain into two nearly finished contigs at >99.9% accuracy. Complex regions with clinically relevant structure were completely resolved. In separate control assemblies on experimental and simulated data for the canonical N16961 cholera reference strain, we obtained 14 scaffolds of greater than 1 kb for the experimental data and 8 scaffolds of greater than 1 kb for the simulated data, which allowed us to correct several errors in contigs assembled from the short-read data alone. This work provides a blueprint for the next generation of rapid microbial identification and full-genome assembly.

The complete methylome of Helicobacter pylori UM032

AbstractBackgroundThe genome of the human gastric pathogen Helicobacter pylori encodes a large number of DNA methyltransferases (MTases), some of which are shared among many strains, and others of which are unique to a given strain. The MTases have potential roles in the survival of the bacterium. In this study, we sequenced a Malaysian H. pylori clinical strain, designated UM032, by using a combination of PacBio Single Molecule, Real-Time (SMRT) and Illumina MiSeq next generation sequencing platforms, and used the SMRT data to characterize the set of methylated bases (the methylome).ResultsThe N4-methylcytosine and N6-methyladenine modifications detected at single-base resolution using SMRT technology revealed 17 methylated sequence motifs corresponding to one Type I and 16 Type II restriction-modification (R-M) systems. Previously unassigned methylation motifs were now assigned to their respective MTases-coding genes. Furthermore, one gene that appears to be inactive in the H. pylori UM032 genome during normal growth was characterized by cloning.ConclusionConsistent with previously-studied H. pylori strains, we show that strain UM032 contains a relatively large number of R-M systems, including some MTase activities with novel specificities. Additional studies are underway to further elucidating the biological significance of the R-M systems in the physiology and pathogenesis of H. pylori.

Draft Genome Sequences of Burkholderia cenocepacia ET12 Lineage Strains K56-2 and BC7

ABSTRACT The Burkholderia cepacia complex (BCC) is a group of closely related bacteria that are responsible for respiratory infections in immunocompromised humans, most notably those with cystic fibrosis (CF). We report the genome sequences for Burkholderia cenocepacia ET12 lineage CF isolates K56-2 and BC7.

Erratum to: Comparing the genomes of Helicobacter pylori

Correction: Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives Yalda Khosravi, Vellaya Rehvathy, Wei Yee Wee, Susana Wang, Primo Baybayan, Siddarth Singh, Meredith Ashby, Junxian Ong, Arlaine Anne Amoyo, Shih Wee Seow, Siew Woh Choo, Tim Perkins, Eng Guan Chua, Alfred Tay, Barry James Marshall, Mun Fai Loke, Khean Lee Goh, Sven Pettersson and Jamuna Vadivelu

Multiple Genome Sequences of Helicobacter pylori Strains of Diverse Disease and Antibiotic Resistance Backgrounds from Malaysia

ABSTRACT Helicobacter pylori causes human gastroduodenal diseases, including chronic gastritis and peptic ulcer disease. It is also a major microbial risk factor for the development of gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Twenty-one strains with different ethnicity, disease, and antimicrobial susceptibility backgrounds were sequenced by use of Illumina HiSeq and PacBio RS platforms.

Comparative genomics reveals the diversity of restriction-modification systems and DNA methylation sites in Listeria monocytogenes

ABSTRACT Listeria monocytogenes is a bacterial pathogen that is found in a wide variety of anthropogenic and natural environments. Genome sequencing technologies are rapidly becoming a powerful tool in facilitating our understanding of how genotype, classification phenotypes, and virulence phenotypes interact to predict the health risks of individual bacterial isolates. Currently, 57 closed L. monocytogenes genomes are publicly available, representing three of the four phylogenetic lineages, and they suggest that L. monocytogenes has high genomic synteny. This study contributes an additional 15 closed L. monocytogenes genomes that were used to determine the associations between the genome and methylome with host invasion magnitude. In contrast to previous findings, large chromosomal inversions and rearrangements were detected in five isolates at the chromosome terminus and within rRNA genes, including a previously undescribed inversion within rRNA-encoding regions. Each isolates epigenome contained highly diverse methyltransferase recognition sites, even within the same serotype and methylation pattern. Eleven strains contained a single chromosomally encoded methyltransferase, one strain contained two methylation systems (one system on a plasmid), and three strains exhibited no methylation, despite the occurrence of methyltransferase genes. In three isolates a new, unknown DNA modification was observed in addition to diverse methylation patterns, accompanied by a novel methylation system. Neither chromosome rearrangement nor strain-specific patterns of epigenome modification observed within virulence genes were correlated with serotype designation, clonal complex, or in vitro infectivity. These data suggest that genome diversity is larger than previously considered in L. monocytogenes and that as more genomes are sequenced, additional structure and methylation novelty will be observed in this organism. IMPORTANCE Listeria monocytogenes is the causative agent of listeriosis, a disease which manifests as gastroenteritis, meningoencephalitis, and abortion. Among Salmonella, Escherichia coli, Campylobacter, and Listeria—causing the most prevalent foodborne illnesses—infection by L. monocytogenes carries the highest mortality rate. The ability of L. monocytogenes to regulate its response to various harsh environments enables its persistence and transmission. Small-scale comparisons of L. monocytogenes focusing solely on genome contents reveal a highly syntenic genome yet fail to address the observed diversity in phenotypic regulation. This study provides a large-scale comparison of 302 L. monocytogenes isolates, revealing the importance of the epigenome and restriction-modification systems as major determinants of L. monocytogenes phylogenetic grouping and subsequent phenotypic expression. Further examination of virulence genes of select outbreak strains reveals an unprecedented diversity in methylation statuses despite high degrees of genome conservation.

Abstract 848: Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer

Introduction Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test detecting blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection. Materials and methods RNA and proteins were isolated from CRC cell line SW480 before and after siRNA-mediated down-modulation of the splicing machinery: SF3B1, U2AF1, and SRSF1. To identify splice variants, mRNA was sequenced (Illumina HiSeq) and analyzed. RNA-seq analysis included quality checks (FASTQ, RSeQC), reads mapping (STAR), differential gene expression (DESeq2) and differential expression of splicing isoforms between conditions (MATS). Results from the in silico analysis were validated by qRT-PCR. Proteins were analyzed by in-depth tandem mass spectrometry (QExactive). A proteogenomic data analysis pipeline was established to enrich the sequence database, against which the mass spectra are searched, with the predicted protein splice variants and identify protein isoforms. To further extend the splice-variant database, PacBio sequencing of full-length transcripts is being performed for the SW480 siSF3B1- and control-samples. Results Differential expression analysis on RNA and protein level proved that the knock-down experiments were performed successfully. The RNA-seq analysis revealed hundreds of mRNA splice variants, including positive controls described in literature. For example, down-modulation of SF3B1 resulted in quantitative mRNA changes of spliced isoforms of ADD3 both in RNA-seq and RT-PCR data. The proteomics experiment yielded over 6000 proteins per sample, among which a number of protein isoforms resulting from alternative splicing. Conclusions and discussion We established a proteogenomic pipeline for the analysis of alternative splicing and provided experimental proof of concept. We expect, however, that the true complexity of RNA variant information remains highly underestimated. We therefore are performing PacBio long-read RNA-seq to validate our approach and to identify additional (novel) splicing events. In future studies we will apply the mRNA-seq based proteogenomic pipeline for detection of protein alterations to a series of adenomas at low- and high-risk of progression, and CRCs to validate the isoforms in a clinically relevant setting. Novel findings will be evaluated for their performance as screening markers for CRC. This work was financially supported by KWF project nr: VU 2013-6025. Citation Format: Malgorzata A. Komor, Annemieke C. Hiemstra, Thang V. Pham, Sander R. Piersma, Robert P. Sebra, Bo W. Han, Meredith Ashby, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond JA Fijneman. Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 848.

Identification of Differentially Expressed Splice Variants by the Proteogenomic Pipeline Splicify

Proteogenomics, i.e. comprehensive integration of genomics and proteomics data, is a powerful approach identifying novel protein biomarkers. This is especially the case for proteins that differ structurally between disease and control conditions. As tumor development is associated with aberrant splicing, we focus on this rich source of cancer specific biomarkers. To this end, we developed a proteogenomic pipeline, Splicify, which can detect differentially expressed protein isoforms. Splicify is based on integrating RNA massive parallel sequencing data and tandem mass spectrometry proteomics data to identify protein isoforms resulting from differential splicing between two conditions. Proof of concept was obtained by applying Splicify to RNA sequencing and mass spectrometry data obtained from colorectal cancer cell line SW480, before and after siRNA-mediated downmodulation of the splicing factors SF3B1 and SRSF1. These analyses revealed 2172 and 149 differentially expressed isoforms, respectively, with peptide confirmation upon knock-down of SF3B1 and SRSF1 compared with their controls. Splice variants identified included RAC1, OSBPL3, MKI67, and SYK. One additional sample was analyzed by PacBio Iso-Seq full-length transcript sequencing after SF3B1 downmodulation. This analysis verified the alternative splicing identified by Splicify and in addition identified novel splicing events that were not represented in the human reference genome annotation. Therefore, Splicify offers a validated proteogenomic data analysis pipeline for identification of disease specific protein biomarkers resulting from mRNA alternative splicing. Splicify is publicly available on GitHub (https://github.com/NKI-TGO/SPLICIFY) and suitable to address basic research questions using pre-clinical model systems as well as translational research questions using patient-derived samples, e.g. allowing to identify clinically relevant biomarkers.

Abstract 2442: Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System

Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 - 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples. Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell. The results highlight the potential for broader application of the Iso-Seq method to more comprehensively characterize alternative splicing in cancer. Citation Format: Meredith Ashby, Ting Hon, Elizabeth Tseng, Aparna Vedula, Tyson A. Clark. Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System [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 2442. doi:10.1158/1538-7445.AM2017-2442

Correction: Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives

Correction: Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives Yalda Khosravi, Vellaya Rehvathy, Wei Yee Wee, Susana Wang, Primo Baybayan, Siddarth Singh, Meredith Ashby, Junxian Ong, Arlaine Anne Amoyo, Shih Wee Seow, Siew Woh Choo, Tim Perkins, Eng Guan Chua, Alfred Tay, Barry James Marshall, Mun Fai Loke, Khean Lee Goh, Sven Pettersson and Jamuna Vadivelu