scholarly journals REscan: inferring repeat expansions and structural variation in paired-end short read sequencing data

2020 ◽  
Author(s):  
Russell Lewis McLaughlin
Keyword(s):  
Structural Variation ◽  
Sequence Data ◽  
Neurological Diseases ◽  
Repeat Expansion ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Repeat Expansions ◽  
Paired End Sequencing

Abstract Motivation Repeat expansions are an important class of genetic variation in neurological diseases. However, the identification of novel repeat expansions using conventional sequencing methods is a challenge due to their typical lengths relative to short sequence reads and difficulty in producing accurate and unique alignments for repetitive sequence. However, this latter property can be harnessed in paired-end sequencing data to infer the possible locations of repeat expansions and other structural variation. Results This article presents REscan, a command-line utility that infers repeat expansion loci from paired-end short read sequencing data by reporting the proportion of reads orientated towards a locus that do not have an adequately mapped mate. A high REscan statistic relative to a population of data suggests a repeat expansion locus for experimental follow-up. This approach is validated using genome sequence data for 259 cases of amyotrophic lateral sclerosis, of which 24 are positive for a large repeat expansion in C9orf72, showing that REscan statistics readily discriminate repeat expansion carriers from non-carriers. Availabilityand implementation C source code at https://github.com/rlmcl/rescan (GNU General Public Licence v3).

scholarly journals Rapid Mycobacterium tuberculosis spoligotyping from uncorrected long reads using Galru

2020 ◽  
Author(s):  
Andrew J. Page ◽  
Nabil-Fareed Alikhan ◽  
Michael Strinden ◽  
Thanh Le Viet ◽  
Timofey Skvortsov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
State Of The Art ◽  
Sequence Data ◽  
Human Pathogen ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Long Reads ◽  
Long Read

AbstractSpoligotyping of Mycobacterium tuberculosis provides a subspecies classification of this major human pathogen. Spoligotypes can be predicted from short read genome sequencing data; however, no methods exist for long read sequence data such as from Nanopore or PacBio. We present a novel software package Galru, which can rapidly detect the spoligotype of a Mycobacterium tuberculosis sample from as little as a single uncorrected long read. It allows for near real-time spoligotyping from long read data as it is being sequenced, giving rapid sample typing. We compare it to the existing state of the art software and find it performs identically to the results obtained from short read sequencing data. Galru is freely available from https://github.com/quadram-institute-bioscience/galru under the GPLv3 open source licence.

scholarly journals Fast and accurate HLA typing from short-read next-generation sequence data with xHLA

2017 ◽  
Vol 114 (30) ◽  
pp. 8059-8064 ◽  
Author(s):  
Chao Xie ◽  
Zhen Xuan Yeo ◽  
Marie Wong ◽  
Jason Piper ◽  
Tao Long ◽  
...  
Keyword(s):  
Sequence Data ◽  
Sequence Similarity ◽  
Amino Acid Level ◽  
Hla Typing ◽  
Sequencing Data ◽  
Desktop Computer ◽  
Short Read ◽  
Short Read Sequencing ◽  
Hla Genes ◽  
Human Chromosome 6

The HLA gene complex on human chromosome 6 is one of the most polymorphic regions in the human genome and contributes in large part to the diversity of the immune system. Accurate typing of HLA genes with short-read sequencing data has historically been difficult due to the sequence similarity between the polymorphic alleles. Here, we introduce an algorithm, xHLA, that iteratively refines the mapping results at the amino acid level to achieve 99–100% four-digit typing accuracy for both class I and II HLA genes, taking only∼3 min to process a 30× whole-genome BAM file on a desktop computer.

scholarly journals Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease

2018 ◽  
Author(s):  
Mark T. W. Ebbert ◽  
Stefan Farrugia ◽  
Jonathon Sens ◽  
Karen Jansen-West ◽  
Tania F. Gendron ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Repeat Expansion ◽  
Whole Genome ◽  
Short Read ◽  
Short Read Sequencing ◽  
Sequencing Technologies ◽  
Long Read ◽  
Repeat Expansions ◽  
Targeted Approach

AbstractBackground: Many neurodegenerative diseases are caused by nucleotide repeat expansions, but most expansions, like the C9orf72 ‘GGGGCC’ (G4C2) repeat that causes approximately 5-7% of all amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases, are too long to sequence using short-read sequencing technologies. It is unclear whether long-read sequencing technologies can traverse these long, challenging repeat expansions. Here, we demonstrate that two long-read sequencing technologies, Pacific Biosciences’ (PacBio) and Oxford Nanopore Technologies’ (ONT), can sequence through disease-causing repeats cloned into plasmids, including the FTD/ALS-causing G4C2 repeat expansion. We also report the first long-read sequencing data characterizing the C9orf72 G4C2 repeat expansion at the nucleotide level in two symptomatic expansion carriers using PacBio whole-genome sequencing and a no-amplification (No-Amp) targeted approach based on CRISPR/Cas9.Results: Both the PacBio and ONT platforms successfully sequenced through the repeat expansions in plasmids. Throughput on the MinlON was a challenge for whole-genome sequencing; we were unable to attain reads covering the human C9orf72 repeat expansion using 15 flow cells. We obtained 8x coverage across the C9orf72 locus using the PacBio Sequel, accurately reporting the unexpanded allele at eight repeats, and reading through the entire expansion with 1324 repeats (7941 nucleotides). Using the No-Amp targeted approach, we attained >800x coverage and were able to identify the unexpanded allele, closely estimate expansion size, and assess nucleotide content in a single experiment. We estimate the individual’s repeat region was >99% G4C2 content, though we cannot rule out small interruptions.Conclusions: Our findings indicate that long-read sequencing is well suited to characterizing known repeat expansions, and for discovering new disease-causing, disease-modifying, or risk-modifying repeat expansions that have gone undetected with conventional short-read sequencing. The PacBio No-Amp targeted approach may have future potential in clinical and genetic counseling environments. Larger and deeper long-read sequencing studies in C9orf72 expansion carriers will be important to determine heterogeneity and whether the repeats are interrupted by non-G4C2 content, potentially mitigating or modifying disease course or age of onset, as interruptions are known to do in other repeat-expansion disorders. These results have broad implications across all diseases where the genetic etiology remains unclear.

scholarly journals Using Short Read Sequencing to Characterise Balanced Reciprocal Translocations in Pigs

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.

scholarly journals ExpansionHunter Denovo: A computational method for locating known and novel repeat expansions in short-read sequencing data

2019 ◽  
Author(s):  
Egor Dolzhenko ◽  
Mark F. Bennett ◽  
Phillip A. Richmond ◽  
Brett Trost ◽  
Sai Chen ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Simulated Data ◽  
Computational Method ◽  
Detection Methods ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Monogenic Disorders ◽  
Genome Wide ◽  
Repeat Expansions

AbstractExpansions of short tandem repeats are responsible for over 40 monogenic disorders, and undoubtedly many more pathogenic repeat expansions (REs) remain to be discovered. Existing methods for detecting REs in short-read sequencing data require predefined repeat catalogs. However recent discoveries have emphasized the need for detection methods that do not require candidate repeats to be specified in advance. To address this need, we introduce ExpansionHunter Denovo, an efficient catalog-free method for genome-wide detection of REs. Analysis of real and simulated data shows that our method can identify large expansions of 41 out of 44 pathogenic repeats, including nine recently reported non-reference REs not discoverable via existing methods.ExpansionHunter Denovo is freely available at https://github.com/Illumina/ExpansionHunterDenovo

scholarly journals Extensive gene duplication in Arabidopsis revealed by pseudo-heterozygosity

2021 ◽  
Author(s):  
Benjamin Jaegle ◽  
Luz Mayela Soto-Jimenez ◽  
Robin Burns ◽  
Fernando A. Rabanal ◽  
Magnus Nordborg
Keyword(s):  
Copy Number ◽  
Structural Variation ◽  
De Novo ◽  
Sequencing Data ◽  
Heterozygous Snps ◽  
Mendelian Segregation ◽  
Short Read ◽  
Short Read Sequencing ◽  
Snp Data ◽  
Number Variation

Background: It is becoming apparent that genomes harbor massive amounts of structural variation, and that this variation has largely gone undetected for technical reasons. In addition to being inherently interesting, structural variation can cause artifacts when short-read sequencing data are mapped to a reference genome. In particular, spurious SNPs (that do not show Mendelian segregation) may result from mapping of reads to duplicated regions. Recalling SNP using the raw reads of the 1001 Arabidopsis Genomes Project we identified 3.3 million heterozygous SNPs (44% of total). Given that Arabidopsis thaliana (A. thaliana) is highly selfing, we hypothesized that these SNPs reflected cryptic copy number variation, and investigated them further. Results: While genuine heterozygosity should occur in tracts within individuals, heterozygosity at a particular locus is instead shared across individuals in a manner that strongly suggests it reflects segregating duplications rather than actual heterozygosity. Focusing on pseudo-heterozygosity in annotated genes, we used GWAS to map the position of the duplicates, identifying 2500 putatively duplicated genes. The results were validated using de novo genome assemblies from six lines. Specific examples included an annotated gene and nearby transposon that, in fact, transpose together. Conclusions: Our study confirms that most heterozygous SNPs calls in A. thaliana are artifacts, and suggest that great caution is needed when analysing SNP data from short-read sequencing. The finding that 10% of annotated genes are copy-number variables, and the realization that neither gene- nor transposon-annotation necessarily tells us what is actually mobile in the genome suggest that future analyses based on independently assembled genomes will be very informative.

scholarly journals Using short read sequencing to characterise balanced reciprocal translocations in pigs

2020 ◽  
Author(s):  
Aniek C. 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.

scholarly journals ExpansionHunter Denovo: a computational method for locating known and novel repeat expansions in short-read sequencing data

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Egor Dolzhenko ◽  
Mark F. Bennett ◽  
Phillip A. Richmond ◽  
Brett Trost ◽  
Sai Chen ◽  
...  
Keyword(s):  
Computational Method ◽  
Sequencing Data ◽  
Short Read ◽  
Short Read Sequencing ◽  
Repeat Expansions

scholarly journals Ultrafast, alignment-free detection of repeat expansions in NGS and RNAseq data

2021 ◽  
Author(s):  
L.G. Fearnley ◽  
M.F. Bennett ◽  
M. Bahlo
Keyword(s):  
Repeat Expansion ◽  
Specific Reference ◽  
Sequencing Data ◽  
Short Read Sequencing ◽  
Dna And Rna ◽  
Rnaseq Data ◽  
Repeat Expansions ◽  
Repetitive Motif ◽  
Genomic Locations ◽  
Short Tandem

AbstractShort tandem repeat expansions are an established cause of diseases such as Huntington’s disease. Bioinformatic methods for detecting repeat expansions in short-read sequencing have revealed new repeat expansions in humans. Current bioinformatic methods to detect repeat expansions require alignment information to identify repetitive motif enrichment at genomic locations. We present superSTR, an ultrafast method that does not require alignment, capable of efficiently processing DNA and RNA sequencing data. superSTR is applied to UK Biobank data to efficiently analyse 49,953 whole exomes in a screening experiment, identifying known mutations as well as diseases not previously associated with REs. superSTR also identifies repeat expansion motifs in RNAseq data, demonstrated in several disorders and cell lines. superSTR is a new tool for the most efficient repeat expansion detection currently possible and complements existing locus-specific, reference dependent repeat expansion analysis tools. superSTR is available from https://github.com/bahlolab/superSTR.

scholarly journals Using short read sequencing to characterise balanced reciprocal translocations in pigs

2020 ◽  
Author(s):  
Aniek C. 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.

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