Insertion variants missing in the human reference genome are widespread among human populations

Abstract Background Structural variants comprise diverse genomic arrangements including deletions, insertions, inversions, and translocations, which can generally be detected in humans through sequence comparison to the reference genome. Among structural variants, insertions are the least frequently identified variants, mainly due to ascertainment bias in the reference genome, lack of previous sequence knowledge, and low complexity of typical insertion sequences. Though recent developments in long-read sequencing deliver promise in annotating individual non-reference insertions, population-level catalogues on non-reference insertion variants have not been identified and the possible functional roles of these hidden variants remain elusive. Results To detect non-reference insertion variants, we developed a pipeline, InserTag, which generates non-reference contigs by local de novo assembly and then infers the full-sequence of insertion variants by tracing contigs from non-human primates and other human genome assemblies. Application of the pipeline to data from 2535 individuals of the 1000 Genomes Project helped identify 1696 non-reference insertion variants and re-classify the variants as retention of ancestral sequences or novel sequence insertions based on the ancestral state. Genotyping of the variants showed that individuals had, on average, 0.92-Mbp sequences missing from the reference genome, 92% of the variants were common (allele frequency > 5%) among human populations, and more than half of the variants were major alleles. Among human populations, African populations were the most divergent and had the most non-reference sequences, which was attributed to the greater prevalence of high-frequency insertion variants. The subsets of insertion variants were in high linkage disequilibrium with phenotype-associated SNPs and showed signals of recent continent-specific selection. Conclusions Non-reference insertion variants represent an important type of genetic variation in the human population, and our developed pipeline, InserTag, provides the frameworks for the detection and genotyping of non-reference sequences missing from human populations.

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Recovery of non-reference sequences missing from the human reference genome

BMC Genomics ◽

10.1186/s12864-019-6107-1 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Ran Li ◽

Xiaomeng Tian ◽

Peng Yang ◽

Yingzhi Fan ◽

Ming Li ◽

...

Keyword(s):

Human Genome ◽

Tandem Repeats ◽

Reference Genome ◽

De Novo ◽

Precise Location ◽

Protein Coding ◽

Human Reference Genome ◽

Mhc Haplotype ◽

Reference Sequences ◽

Flanking Regions

Abstract Background The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Results Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6113 NRS adding up to 12.8 Mb. Besides 1571 insertions, we detected 3041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Conclusions Our study detected a large number of NRS including many alternate alleles which are previously uncharacterized. We suggested that the origin of alternate alleles was associated with tandem repeats. Our results enriched the spectrum of genetic variations in human genome.

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Towards a reference genome that captures global genetic diversity

Nature Communications ◽

10.1038/s41467-020-19311-w ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Karen H. Y. Wong ◽

Walfred Ma ◽

Chun-Yu Wei ◽

Erh-Chan Yeh ◽

Wan-Jia Lin ◽

...

Keyword(s):

Genetic Diversity ◽

Reference Genome ◽

Regulatory Elements ◽

Human Populations ◽

Single Individual ◽

Rna Seq ◽

Human Reference Genome ◽

Reference Sequences ◽

Genome Annotations ◽

Unmapped Reads

Abstract The current human reference genome is predominantly derived from a single individual and it does not adequately reflect human genetic diversity. Here, we analyze 338 high-quality human assemblies of genetically divergent human populations to identify missing sequences in the human reference genome with breakpoint resolution. We identify 127,727 recurrent non-reference unique insertions spanning 18,048,877 bp, some of which disrupt exons and known regulatory elements. To improve genome annotations, we linearly integrate these sequences into the chromosomal assemblies and construct a Human Diversity Reference. Leveraging this reference, an average of 402,573 previously unmapped reads can be recovered for a given genome sequenced to ~40X coverage. Transcriptomic diversity among these non-reference sequences can also be directly assessed. We successfully map tens of thousands of previously discarded RNA-Seq reads to this reference and identify transcription evidence in 4781 gene loci, underlining the importance of these non-reference sequences in functional genomics. Our extensive datasets are important advances toward a comprehensive reference representation of global human genetic diversity.

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Rapid low-cost assembly of the Drosophila melanogaster reference genome using low-coverage, long-read sequencing

10.1101/267401 ◽

2018 ◽

Cited By ~ 6

Author(s):

Edwin A. Solares ◽

Mahul Chakraborty ◽

Danny E. Miller ◽

Shannon Kalsow ◽

Kate Hall ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Genetic Variation ◽

Large Scale ◽

Reference Genome ◽

De Novo ◽

Low Cost ◽

Nucleotide Polymorphisms ◽

Structural Variants ◽

High Coverage ◽

Reference Assembly

ABSTRACTAccurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from large-scale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using high-coverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hours. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD).

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Recovery of non-reference sequences missing from the human reference genome

10.21203/rs.2.11742/v1 ◽

2019 ◽

Author(s):

Ran Li ◽

Xiaomeng Tian ◽

Peng Yang ◽

Yingzhi Fan ◽

Ming Li ◽

...

Keyword(s):

Tandem Repeats ◽

Reference Genome ◽

De Novo ◽

Precise Location ◽

Protein Coding ◽

Human Reference Genome ◽

Protein Coding Genes ◽

Mhc Haplotype ◽

Reference Sequences ◽

Flanking Regions

Abstract The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6,113 NRS adding up to 12.8 Mb. Besides 1,571 insertions, we detected 3,041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1,143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Our study enriched the spectrum of human genetic variations.

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Recovery of non-reference sequences missing from the human reference genome

10.21203/rs.2.11742/v2 ◽

2019 ◽

Author(s):

Ran Li ◽

Xiaomeng Tian ◽

Peng Yang ◽

Yingzhi Fan ◽

Ming Li ◽

...

Keyword(s):

Human Genome ◽

Tandem Repeats ◽

Reference Genome ◽

De Novo ◽

Precise Location ◽

Protein Coding ◽

Human Reference Genome ◽

Mhc Haplotype ◽

Reference Sequences ◽

Flanking Regions

Abstract Background The non-reference sequences (NRS) represent structure variations in human genome with potential functional significance. However, besides the known insertions, it is currently unknown whether other types of structure variations with NRS exist. Results Here, we compared 31 human de novo assemblies with the current reference genome to identify the NRS and their location. We resolved the precise location of 6,113 NRS adding up to 12.8 Mb. Besides 1,571 insertions, we detected 3,041 alternate alleles, which were defined as having less than 90% (or none) identity with the reference alleles. These alternate alleles overlapped with 1,143 protein-coding genes including a putative novel MHC haplotype. Further, we demonstrated that the alternate alleles and their flanking regions had high content of tandem repeats, indicating that their origin was associated with tandem repeats. Conclusions Our study detected a large number of NRS including many alternate alleles which are previously uncharacterized. We suggested that the origin of alternate alleles was associated with tandem repeats. Our results enriched the spectrum of genetic variations in human genome.

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2-kupl: mapping-free variant detection from DNA-seq data of matched samples

BMC Bioinformatics ◽

10.1186/s12859-021-04185-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Yunfeng Wang ◽

Haoliang Xue ◽

Christine Pourcel ◽

Yang Du ◽

Daniel Gautheret

Keyword(s):

Dna Sequences ◽

Reference Genome ◽

Point Mutations ◽

Variant Calling ◽

Low Complexity ◽

Structural Variants ◽

Sequencing Data ◽

Bacterial Strains ◽

Two Samples ◽

Variant Detection

Abstract Background The detection of genome variants, including point mutations, indels and structural variants, is a fundamental and challenging computational problem. We address here the problem of variant detection between two deep-sequencing (DNA-seq) samples, such as two human samples from an individual patient, or two samples from distinct bacterial strains. The preferred strategy in such a case is to align each sample to a common reference genome, collect all variants and compare these variants between samples. Such mapping-based protocols have several limitations. DNA sequences with large indels, aggregated mutations and structural variants are hard to map to the reference. Furthermore, DNA sequences cannot be mapped reliably to genomic low complexity regions and repeats. Results We introduce 2-kupl, a k-mer based, mapping-free protocol to detect variants between two DNA-seq samples. On simulated and actual data, 2-kupl achieves higher accuracy than other mapping-free protocols. Applying 2-kupl to prostate cancer whole exome sequencing data, we identify a number of candidate variants in hard-to-map regions and propose potential novel recurrent variants in this disease. Conclusions We developed a mapping-free protocol for variant calling between matched DNA-seq samples. Our protocol is suitable for variant detection in unmappable genome regions or in the absence of a reference genome.

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Establishment of an eHAP1 Human Haploid Cell Line Hybrid Reference Genome Assembled from Short and Long Reads

10.1101/822593 ◽

2019 ◽

Author(s):

William D. Law ◽

René L. Warren ◽

Andrew S. McCallion

Keyword(s):

Cell Line ◽

Reference Genome ◽

De Novo ◽

Open Chromatin ◽

Haploid Cell ◽

Structural Variants ◽

High Quality ◽

Short Reads ◽

Long Reads ◽

Low Coverage

AbstractBackgroundHaploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies.ResultsWe generated ~15x coverage Nanopore long reads from ten GridION flowcells. We utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions.ConclusionsBy integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. We identify structural variants using long reads, including some that may impact putative regulatory elements. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line.

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Fast and accurate reference-guided scaffolding of draft genomes

10.1101/519637 ◽

2019 ◽

Cited By ~ 13

Author(s):

Michael Alonge ◽

Sebastian Soyk ◽

Srividya Ramakrishnan ◽

Xingang Wang ◽

Sara Goodwin ◽

...

Keyword(s):

Open Source ◽

Genome Analysis ◽

Reference Genome ◽

De Novo ◽

Genetic Maps ◽

Alternative Methods ◽

Structural Variants ◽

Pan Genome ◽

Long Read ◽

Increasing Demand

AbstractBackgroundAs the number of new genome assemblies continues to grow, there is increasing demand for methods to coalesce contigs from draft assemblies into pseudomolecules. Most current methods use genetic maps, optical maps, chromatin conformation (Hi-C), or other long-range linking data, however these data are expensive and analysis methods often fail to accurately order and orient a high percentage of assembly contigs. Other approaches utilize alignments to a reference genome for ordering and orienting, however these tools rely on slow aligners and are not robust to repetitive contigs.ResultsWe present RaGOO, an open-source reference-guided contig ordering and orienting tool that leverages the speed and sensitivity of Minimap2 to accurately achieve chromosome-scale assemblies in just minutes. With the pseudomolecules constructed, RaGOO identifies structural variants, including those spanning sequencing gaps that are not reported by alternative methods. We show that RaGOO accurately orders and orients contigs into nearly complete chromosomes based on de novo assemblies of Oxford Nanopore long-read sequencing from three wild and domesticated tomato genotypes, including the widely used M82 reference cultivar. We then demonstrate the scalability and utility of RaGOO with a pan-genome analysis of 103 Arabidopsis thaliana accessions by examining the structural variants detected in the newly assembled pseudomolecules. RaGOO is available open-source with an MIT license at https://github.com/malonge/RaGOO.ConclusionsWe demonstrate that with a highly contiguous assembly and a structurally accurate reference genome, reference-guided scaffolding with RaGOO outperforms error-prone reference-free methods and enable rapid pan-genome analysis.

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A diploid assembly-based benchmark for variants in the major histocompatibility complex

Nature Communications ◽

10.1038/s41467-020-18564-9 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 2

Author(s):

Chen-Shan Chin ◽

Justin Wagner ◽

Qiandong Zeng ◽

Erik Garrison ◽

Shilpa Garg ◽

...

Keyword(s):

Major Histocompatibility Complex ◽

Performance Assessment ◽

Reference Genome ◽

De Novo ◽

Major Histocompatibility ◽

Structural Variants ◽

Histocompatibility Complex ◽

Human Genomes ◽

Long Reads ◽

Complex Variation

Abstract Most human genomes are characterized by aligning individual reads to the reference genome, but accurate long reads and linked reads now enable us to construct accurate, phased de novo assemblies. We focus on a medically important, highly variable, 5 million base-pair (bp) region where diploid assembly is particularly useful - the Major Histocompatibility Complex (MHC). Here, we develop a human genome benchmark derived from a diploid assembly for the openly-consented Genome in a Bottle sample HG002. We assemble a single contig for each haplotype, align them to the reference, call phased small and structural variants, and define a small variant benchmark for the MHC, covering 94% of the MHC and 22368 variants smaller than 50 bp, 49% more variants than a mapping-based benchmark. This benchmark reliably identifies errors in mapping-based callsets, and enables performance assessment in regions with much denser, complex variation than regions covered by previous benchmarks.

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Assemblytics: a web analytics tool for the detection of assembly-based variants

10.1101/044925 ◽

2016 ◽

Cited By ~ 5

Author(s):

Maria Nattestad ◽

Michael C Schatz

Keyword(s):

Genome Assembly ◽

Related Species ◽

Reference Genome ◽

De Novo ◽

Web Analytics ◽

Structural Variants ◽

De Novo Genome Assembly ◽

Interactive Visualizations ◽

Web App ◽

Filtering Approach

Summary: Assemblytics is a web app for detecting and analyzing structural variants from a de novo genome assembly aligned to a reference genome. It incorporates a unique anchor filtering approach to increase robustness to repetitive elements, and identifies six classes of variants based on their distinct alignment signatures. Assemblytics can be applied both to comparing aberrant genomes, such as human cancers, to a reference, or to identify differences between related species. Multiple interactive visualizations enable in-depth explorations of the genomic distributions of variants. Availability and Implementation: http://qb.cshl.edu/assemblytics, https://github.com/marianattestad/assemblytics Contact: [email protected]

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