A Family-Based Probabilistic Method for Capturing De Novo Mutations from High-Throughput Short-Read Sequencing Data

Variation within human genomes is distributed unevenly and variants show spatial clustering. DNA-replication related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. We reanalyzed haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments. We found local template switching to explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations with a small number appearing as de novo mutations. We developed computational tools for genotyping candidate template switch loci using short-read sequencing data and for identification of template switch events using both short-read data and genotype data. These tools will enable building a catalogue of affected loci and studying the cellular mechanisms behind template switching both in healthy organisms and in disease. Strikingly, we noticed that widely-used analysis pipelines for short-read sequencing data - capable of identifying single nucleotide changes - may miss TSM-origin inversions of tens of base pairs, potentially invalidating medical genetic studies searching for causative alleles behind genetic diseases.

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Optimization of de novo transcriptome assembly from high-throughput short read sequencing data improves functional annotation for non-model organisms

BMC Bioinformatics ◽

10.1186/1471-2105-13-170 ◽

2012 ◽

Vol 13 (1) ◽

pp. 170 ◽

Cited By ~ 24

Author(s):

Berat Z Haznedaroglu ◽

Darryl Reeves ◽

Hamid Rismani-Yazdi ◽

Jordan Peccia

Keyword(s):

High Throughput ◽

Functional Annotation ◽

De Novo ◽

Transcriptome Assembly ◽

Model Organisms ◽

De Novo Transcriptome Assembly ◽

Sequencing Data ◽

De Novo Transcriptome ◽

Short Read ◽

Short Read Sequencing

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Indel variant analysis of short-read sequencing data with Scalpel

10.1101/028050 ◽

2015 ◽

Cited By ~ 1

Author(s):

Han Fang ◽

Ewa A. Grabowska ◽

Kanika Arora ◽

Vladimir Vacic ◽

Michael C. Zody ◽

...

Keyword(s):

Large Scale ◽

De Novo ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Indel Detection ◽

Indel Calling ◽

Assembly Technique ◽

Variant Analysis ◽

Family Based

As the second most common type of variations in the human genome, insertions and deletions (indels) have been linked to many diseases, but indels of more than a few bases are still challenging to discover from short-read sequencing data. Scalpel (http://scalpel.sourceforge.net) is open-source software for reliable indel detection based on the micro-assembly technique. To date, it has been successfully used to discover mutations in novel candidate genes for autism, and is extensively used in other large-scale studies of human diseases. This protocol gives an overview of the algorithm and describes how to use Scalpel to perform highly accurate indel calling from whole genome and exome sequencing data. We provide detailed instructions for an exemplary family-based de novo study, but we also characterize the other two supported modes of operation for single sample and somatic analysis. Indel normalization, visualization, and annotation of the mutations are also illustrated. Using a standard server, indel discovery and characterization in the exonic regions of the example sequencing data can be finished in ~6 hours after read mapping.

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DACCOR - Detection, charACterization, and reconstruction of Repetitive regions in bacterial genomes

10.7287/peerj.preprints.3480v1 ◽

2017 ◽

Author(s):

Alexander Seitz ◽

Friederike Hanssen ◽

Kay Nieselt

Keyword(s):

Base Pair ◽

Repetitive Sequence ◽

Reference Genome ◽

De Novo ◽

Treponema Pallidum ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Reads ◽

Short Read Sequencing

The reconstruction of genomes using mapping based approaches with short reads experiences difficulties when resolving repetitive regions. These repetitive regions in genomes result in low mapping qualities of the respective reads, which in turn lead to many unresolved bases of the genotypers. Currently, the reconstruction of these regions is often based on modified references in which the repetitive regions are masked. However, for many references such masked genomes are not available or are based on repetitive regions of other genomes. Our idea is to identify repetitive regions in the reference genome de novo. These regions can then be used to reconstruct them separately using short read sequencing data. Afterwards the reconstructed repetitive sequence can be inserted into the reconstructed genome. We present the program DACCOR, which performs these steps automatically. Our results show an increased base pair resolution of the repetitive regions in the reconstruction of Treponema pallidum samples, resulting in fewer unresolved bases.

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High-throughput Interpretation of Killer-cell Immunoglobulin-like Receptor Short-read Sequencing Data with PING

10.1101/2021.03.24.436770 ◽

2021 ◽

Author(s):

Wesley Marin ◽

Ravi Dandekar ◽

Danillo G. Augusto ◽

Tasneem Yusufali ◽

Bianca Heyn ◽

...

Keyword(s):

High Resolution ◽

High Throughput ◽

Copy Number ◽

Killer Cell ◽

Gene Copy Number ◽

Gene Copy ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Kir Genes

The killer-cell immunoglobulin-like receptor ( KIR) complex on chromosome 19 encodes receptors that modulate the activity of natural killer cells, and variation in these genes has been linked to infectious and autoimmune disease, as well as having bearing on pregnancy and transplant outcomes. The medical relevance and high variability of KIR genes makes short-read sequencing an attractive technology for interrogating the region, providing a high-throughput, high-fidelity sequencing method that is cost-effective. However, because this gene complex is characterized by extensive nucleotide polymorphism, structural variation including gene fusions and deletions, and a high level of homology between genes, its interrogation at high resolution has been thwarted by bioinformatic challenges, with most studies limited to examining presence or absence of specific genes. Here, we present the PING (Pushing Immunogenetics to the Next Generation) pipeline, which incorporates empirical data, novel alignment strategies and a custom alignment processing workflow to enable high-throughput KIR sequence analysis from short-read data. PING provides KIR gene copy number classification functionality for all KIR genes through use of a comprehensive alignment reference. The gene copy number determined per individual enables an innovative genotype determination workflow using genotype-matched references. Together, these methods address the challenges imposed by the structural complexity and overall homology of the KIR complex. To determine copy number and genotype determination accuracy, we applied PING to European and African validation cohorts and a synthetic dataset. PING demonstrated exceptional copy number determination performance across all datasets and robust genotype determination performance. Finally, an investigation into discordant genotypes for the synthetic dataset provides insight into misaligned reads, advancing our understanding in interpretation of short-read sequencing data in complex genomic regions. PING promises to support a new era of studies of KIR polymorphism, delivering high-resolution KIR genotypes that are highly accurate, enabling high-quality, high-throughput KIR genotyping for disease and population studies.

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A framework for the detection of de novo mutations in family-based sequencing data

European Journal of Human Genetics ◽

10.1038/ejhg.2016.147 ◽

2016 ◽

Vol 25 (2) ◽

pp. 227-233 ◽

Cited By ~ 19

Author(s):

Laurent C Francioli ◽

◽

Mircea Cretu-Stancu ◽

Kiran V Garimella ◽

Menachem Fromer ◽

...

Keyword(s):

De Novo ◽

Sequencing Data ◽

De Novo Mutations ◽

Family Based

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Extensive gene duplication in Arabidopsis revealed by pseudo-heterozygosity

10.1101/2021.11.15.468652 ◽

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.

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DACCOR–Detection, characterization, and reconstruction of repetitive regions in bacterial genomes

PeerJ ◽

10.7717/peerj.4742 ◽

2018 ◽

Vol 6 ◽

pp. e4742 ◽

Cited By ~ 1

Author(s):

Alexander Seitz ◽

Friederike Hanssen ◽

Kay Nieselt

Keyword(s):

Base Pair ◽

Repetitive Sequence ◽

Reference Genome ◽

De Novo ◽

Treponema Pallidum ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Reads ◽

Short Read Sequencing

The reconstruction of genomes using mapping-based approaches with short reads experiences difficulties when resolving repetitive regions. These repetitive regions in genomes result in low mapping qualities of the respective reads, which in turn lead to many unresolved bases. Currently, the reconstruction of these regions is often based on modified references in which the repetitive regions are masked. However, for many references, such masked genomes are not available or are based on repetitive regions of other genomes. Our idea is to identify repetitive regions in the reference genome de novo. These regions can then be used to reconstruct them separately using short read sequencing data. Afterward, the reconstructed repetitive sequence can be inserted into the reconstructed genome. We present the program detection, characterization, and reconstruction of repetitive regions, which performs these steps automatically. Our results show an increased base pair resolution of the repetitive regions in the reconstruction of Treponema pallidum samples, resulting in fewer unresolved bases.

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Illumina short-read sequencing data, de novo assembly and annotations of the Drosophila nasuta nasuta genome

Data in Brief ◽

10.1016/j.dib.2020.106674 ◽

2021 ◽

Vol 34 ◽

pp. 106674

Author(s):

Stafny DSouza ◽

Koushik Ponnanna ◽

Amruthavalli Chokkanna ◽

Nallur Ramachandra

Keyword(s):

De Novo Assembly ◽

De Novo ◽

Sequencing Data ◽

Short Read ◽

Short Read Sequencing ◽

Drosophila Nasuta ◽

Drosophila Nasuta Nasuta

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A high-throughput multiplexing and selection strategy to complete bacterial genomes

GigaScience ◽

10.1093/gigascience/giab079 ◽

2021 ◽

Vol 10 (12) ◽

Cited By ~ 1

Author(s):

Sergio Arredondo-Alonso ◽

Anna K Pöntinen ◽

François Cléon ◽

Rebecca A Gladstone ◽

Anita C Schürch ◽

...

Keyword(s):

High Throughput ◽

Selection Strategy ◽

Bacterial Isolates ◽

Sequencing Data ◽

Bacterial Genomes ◽

Short Read ◽

Short Read Sequencing ◽

Long Read ◽

Isolate Selection ◽

Selection Of

Abstract Background Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.

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