Faculty Opinions recommendation of Paragraph: a graph-based structural variant genotyper for short-read sequence data.

Background Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, direct from paired-end short read data. Results ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 96% of known IS insertions in the analysis of simulated reads, and 98% in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was reliable for average genome-wide read depths >20x. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots. Conclusions ISMapper provides a rapid and robust method for identifying IS insertion sites direct from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.

Download Full-text

Mutational sequencing for accurate count and long-range assembly

10.1101/149740 ◽

2017 ◽

Author(s):

Vijay Kumar ◽

Julie Rosenbaum ◽

Zihua Wang ◽

Talitha Forcier ◽

Michael Ronemus ◽

...

Keyword(s):

Long Range ◽

Copy Number ◽

Sequence Data ◽

Template Molecule ◽

Short Read ◽

Unique Pattern ◽

Short Read Sequence

ABSTRACTWe introduce a new protocol, mutational sequencing or muSeq, which randomly deaminates unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling a 135,000 fragment PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone.

Download Full-text

MOST: a modified MLST typing tool based on short read sequencing

PeerJ ◽

10.7717/peerj.2308 ◽

2016 ◽

Vol 4 ◽

pp. e2308 ◽

Cited By ~ 63

Author(s):

Rediat Tewolde ◽

Timothy Dallman ◽

Ulf Schaefer ◽

Carmen L. Sheppard ◽

Philip Ashton ◽

...

Keyword(s):

Conventional Method ◽

Sequence Data ◽

Pcr Amplification ◽

Housekeeping Genes ◽

Data Sets ◽

Bacterial Genomes ◽

Bacterial Populations ◽

Short Read ◽

Short Read Sequence ◽

Low Coverage

Multilocus sequence typing (MLST) is an effective method to describe bacterial populations. Conventionally, MLST involves Polymerase Chain Reaction (PCR) amplification of housekeeping genes followed by Sanger DNA sequencing. Public Health England (PHE) is in the process of replacing the conventional MLST methodology with a method based on short read sequence data derived from Whole Genome Sequencing (WGS). This paper reports the comparison of the reliability of MLST results derived from WGS data, comparing mapping and assembly-based approaches to conventional methods using 323 bacterial genomes of diverse species. The sensitivity of the two WGS based methods were further investigated with 26 mixed and 29 low coverage genomic data sets fromSalmonella enteridisandStreptococcus pneumoniae. Of the 323 samples, 92.9% (n= 300), 97.5% (n= 315) and 99.7% (n= 322) full MLST profiles were derived by the conventional method, assembly- and mapping-based approaches, respectively. The concordance between samples that were typed by conventional (92.9%) and both WGS methods was 100%. From the 55 mixed and low coverage genomes, 89.1% (n= 49) and 67.3% (n= 37) full MLST profiles were derived from the mapping and assembly based approaches, respectively. In conclusion, deriving MLST from WGS data is more sensitive than the conventional method. When comparing WGS based methods, the mapping based approach was the most sensitive. In addition, the mapping based approach described here derives quality metrics, which are difficult to determine quantitatively using conventional and WGS-assembly based approaches.

Download Full-text

Using Short Read Sequencing to Characterise Balanced Reciprocal Translocations in Pigs

10.21203/rs.3.rs-28830/v1 ◽

2020 ◽

Author(s):

Aniek Cornelia Bouwman ◽

Martijn F.L. Derks ◽

Marleen L.W.J. Broekhuijse ◽

Barbara Harlizius ◽

Roel F. Veerkamp

Keyword(s):

Sequence Data ◽

Variant Calling ◽

Reciprocal Translocations ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Short Read Sequence ◽

Staining Techniques ◽

Chromosome Staining ◽

Paired End Sequencing

Abstract Background A balanced constitutional reciprocal translocation (RT) is a mutual exchange of terminal segments of two non-homologous chromosomes without any loss or gain of DNA in germline cells. Carriers of balanced RTs are viable individuals with no apparent phenotypical consequences. These animals produce, however, unbalanced gametes and show therefore reduced fertility and offspring with congenital abnormalities. This cytogenetic abnormality is usually detected using chromosome staining techniques. The aim of this study was to test the possibilities of using paired end short read sequencing for detection of balanced RTs in boars and investigate their breakpoints and junctions.Results Balanced RTs were recovered in a blinded analysis, using structural variant calling software DELLY, in 6 of the 7 carriers with 30 fold short read paired end sequencing. In 15 non-carriers we did not detect any RTs. Reducing the coverage to 20 fold, 15 fold and 10 fold showed that at least 20 fold coverage is required to obtain good results. One RT was not detected using the blind screening, however, a highly likely RT was discovered after unblinding. This RT was located in a repetitive region, showing the limitations of short read sequence data. The detailed analysis of the breakpoints and junctions suggested three junctions showing microhomology, three junctions with blunt-end ligation, and three micro-insertions at the breakpoint junctions. The RTs detected also showed to disrupt genes.Conclusions We conclude that paired end short read sequence data can be used to detect and characterize balanced reciprocal translocations, if sequencing depth is at least 20 fold coverage. However, translocations in repetitive areas may require large fragments or even long read sequence data.

Download Full-text

Rapid, Paralog-Sensitive CNV Analysis of 2457 Human Genomes Using QuicK-mer2

Genes ◽

10.3390/genes11020141 ◽

2020 ◽

Vol 11 (2) ◽

pp. 141 ◽

Cited By ~ 5

Author(s):

Feichen Shen ◽

Jeffrey M. Kidd

Keyword(s):

Copy Number Variation ◽

Copy Number ◽

Sequence Data ◽

Data Sets ◽

Short Read ◽

Major Mechanism ◽

Rapid Construction ◽

A Genome ◽

Number Variation ◽

Short Read Sequence

Gene duplication is a major mechanism for the evolution of gene novelty, and copy-number variation makes a major contribution to inter-individual genetic diversity. However, most approaches for studying copy-number variation rely upon uniquely mapping reads to a genome reference and are unable to distinguish among duplicated sequences. Specialized approaches to interrogate specific paralogs are comparatively slow and have a high degree of computational complexity, limiting their effective application to emerging population-scale data sets. We present QuicK-mer2, a self-contained, mapping-free approach that enables the rapid construction of paralog-specific copy-number maps from short-read sequence data. This approach is based on the tabulation of unique k-mer sequences from short-read data sets, and is able to analyze a 20X coverage human genome in approximately 20 min. We applied our approach to newly released sequence data from the 1000 Genomes Project, constructed paralog-specific copy-number maps from 2457 unrelated individuals, and uncovered copy-number variation of paralogous genes. We identify nine genes where none of the analyzed samples have a copy number of two, 92 genes where the majority of samples have a copy number other than two, and describe rare copy number variation effecting multiple genes at the APOBEC3 locus.

Download Full-text

Evaluating short-read sequence data from the highly redundant, novel transcriptome of Polarella glacialis

Genome Biology ◽

10.1186/gb-2011-12-s1-p5 ◽

2011 ◽

Vol 12 (Suppl 1) ◽

pp. P5

Author(s):

Theodore R Gibbons ◽

Gregory T Concepcion ◽

Tsvetan R Bachvaroff ◽

Charles F Delwiche

Keyword(s):

Sequence Data ◽

Short Read ◽

Short Read Sequence

Download Full-text

De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae

Genome Research ◽

10.1101/gr.083311.108 ◽

2008 ◽

Vol 19 (2) ◽

pp. 294-305 ◽

Cited By ~ 103

Author(s):

J. A. Reinhardt ◽

D. A. Baltrus ◽

M. T. Nishimura ◽

W. R. Jeck ◽

C. D. Jones ◽

...

Keyword(s):

Pseudomonas Syringae ◽

De Novo Assembly ◽

De Novo ◽

Sequence Data ◽

Short Read ◽

Short Read Sequence ◽

Low Coverage

Download Full-text

Prioritisation of Structural Variant Calls in Cancer Genomes

10.1101/084640 ◽

2016 ◽

Author(s):

Miika J Ahdesmäki ◽

Brad Chapman ◽

Pablo E Cingolani ◽

Oliver Hofmann ◽

Aleksandr Sidoruk ◽

...

Keyword(s):

Dna Sequence ◽

Gene Fusion ◽

Sequence Data ◽

High Impact ◽

Structural Variants ◽

Short Read ◽

Structural Variant ◽

Dna Sequence Data ◽

Cancer Genomes ◽

Fusion Detection

AbstractSensitivity of short read DNA-sequencing for gene fusion detection is improving, but is hampered by the significant amount of noise composed of uninteresting or false positive hits in the data. In this paper we describe a tiered prioritisation approach to extract high impact gene fusion events. Using cell line and patient DNA sequence data we improve the annotation and interpretation of structural variant calls to best highlight likely cancer driving fusions. We also considerably improve on the automated visualisation of the high impact structural variants to highlight the effects of the variants on the resulting transcripts. The resulting framework greatly improves on readily detecting clinically actionable structural variants.

Download Full-text