Pitfalls of haplotype phasing from amplicon-based long-read sequencing

Laver, Thomas W; Caswell, Richard; Moore, Karen; Poschmann, Jérémie; Johnson, Matthew; Owens, Martina; Ellard, Sian; Paszkiewicz, Konrad; Weedon, Michael N.

doi:10.1038/srep21746

Cited by 70 publications

(65 citation statements)

References 24 publications

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“…A previous study reports a chimeric arrangement frequency of 6.5% for 29 cycles of amplification for mutations 9kbp apart. 22 Chimeric reads may account for the very few discordant reads in some of our samples, as shown in Table 2. Nonetheless, these were so minor in comparison to the great majority of the reads that phase could be unequivocally determined.…”

Section: Establishing Phasementioning

confidence: 92%

“…The generation of chimeric reads is known to interfere with amplicon-based haplotype phasing by switching a variant from one strand to another, potentially generating new haplotypes not present in the original sample. 22 In practice, chimeric reads randomize the arrangement of the somatic and germline mutations. The frequency of chimeric arrangements is highly dependent on the distance between mutations and the number of PCR cycles used to make the amplicons.…”

Section: Establishing Phasementioning

confidence: 99%

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Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Biallelic Loss of ENG or ACVRL1

Snellings

Gallione

Clark

et al. 2019

Preprint

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Hereditary Hemorrhagic Telangiectasia (HHT) is a Mendelian disease characterized by vascular malformations including visceral arteriovenous malformations and mucosal telangiectasia. HHT is caused by loss-of-function mutations in one of 3 genes; ENG, ACVRL1 or SMAD4 and is inherited as an autosomal dominant condition. Intriguingly, the constitutional mutation causing HHT is present throughout the body, yet the multiple vascular malformations in HHT patients occur focally, rather than manifesting as a systemic vascular defect. This disconnect between genotype and phenotype suggests that a local event is necessary for the development of vascular malformations. We investigated the hypothesis that local somatic mutations seed the formation HHTrelated telangiectasia in a genetic two-hit mechanism. We identified low-frequency somatic mutations in 9/19 telangiectasia using high depth next-generation sequencing. We established phase for 7 of 9 samples using long-read sequencing, which confirm that the germline and somatic mutations in all 7 samples exist in trans configuration; consistent with a genetic two-hit mechanism. These combined data suggest that biallelic loss of ENG or ACVRL1 may be a required event in the development of telangiectasia, and that rather than haploinsufficiency, vascular malformations in HHT are caused by a Knudsonian two-hit mechanism.

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Section: Establishing Phasementioning

confidence: 92%

Section: Establishing Phasementioning

confidence: 99%

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Biallelic Loss of ENG or ACVRL1

Snellings

Gallione

Clark

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Long reads allow for the phased genotyping on polyploid samples, eliminating the need for performing paired-end sequencing with large insert sizes to resolve the gametic phase of distant genetic polymorphisms [13,14].…”

Section: Resultsmentioning

confidence: 99%

“…This, however was made extremely difficult due to PCR chimera formation, most likely during the second barcoding PCR, reported already in recent papers [13,14]. Partly related to the unique ability of using long strands, PCR chimeras are formed when the complementary strand synthesis is incomplete or when the PCR enzyme complex switches template strands during the amplification.…”

Section: Phasingmentioning

confidence: 99%

Beyond Homopolymer Errors: a Systematic Investigation of Nanopore-based DNA Sequencing Characteristics Using HLA-DQA2

Sarkozy

Molnár

Fogl

et al. 2017

Period. Polytech. Elec. Eng. Comp. Sci.

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“…The formation of artificial chimeras during amplification is an important source of artefacts in amplicon sequencing projects (Lenz & Becker, 2008;Smyth et al, 2010), including those with newer sequencing technologies (Laver et al, 2016). Chimeras are challenging to identify as artefacts because they resemble real alleles generated by recombination, particularly in multigene systems under high rates of interlocus genetic exchange ("concerted evolution"), which is common in many MHC systems (Reto et al, 2008;Reto et al, 2010;Edwards, Grahn, & Potts, 1995;Gillingham et al, 2016;Hess & Edwards, 2002;Wittzell et al, 1999).…”

Section: Chimeras In Genotyping Pipelinesmentioning

confidence: 99%

A novel workflow to improve multi-locus genotyping of wildlife species: an experimental set-up with a known model system

Gillingham

Montero

Wilhelm

et al. 2019

Preprint

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Genotyping novel complex multigene systems is particularly challenging in non-model organisms.Target primers frequently amplify simultaneously multiple loci leading to high PCR and sequencing artefacts such as chimeras and allele amplification bias. Most next-generation sequencing genotyping pipelines have been validated in non-model systems whereby the real genotype is unknown and the generation of artefacts may be highly repeatable. Further hindering accurate genotyping, the relationship between artefacts and copy number variation (CNV) within a PCR remains poorly described. Here we investigate the latter by experimentally combining multiple known major histocompatibility complex (MHC) haplotypes of a model organism (chicken, Gallus gallus, 43 artificial genotypes with 2-13 alleles per amplicon). In addition to well defined "optimal" primers, we simulated a non-model species situation by designing "naïve" primers, with sequence data from closely related Galliform species. We applied a novel open-source genotyping pipeline (ACACIA) to the data, and compared its performance with another, previously published, pipeline.ACACIA yielded very high allele calling accuracy (>98%). Non-chimeric artefacts increased linearly with increasing CNV but chimeric artefacts leveled when amplifying more than 4-6 alleles.As expected, we found heterogeneous amplification efficiency of allelic variants when coamplifying multiple loci. Using our validated ACACIA pipeline and the example data of this study, we discuss in detail the pitfalls researchers should avoid in order to reliably genotype complex multigene systems. ACACIA and the datasets used in this study are publicly available at GitLab and FigShare(https://gitlab.com/psc_santos/ACACIA and https://figshare.com/projects/ACACIA/66485).

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Pitfalls of haplotype phasing from amplicon-based long-read sequencing

Cited by 70 publications

References 24 publications

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Biallelic Loss of ENG or ACVRL1

Somatic Mutations in Vascular Malformations of Hereditary Hemorrhagic Telangiectasia Result in Biallelic Loss of ENG or ACVRL1

Beyond Homopolymer Errors: a Systematic Investigation of Nanopore-based DNA Sequencing Characteristics Using HLA-DQA2

A novel workflow to improve multi-locus genotyping of wildlife species: an experimental set-up with a known model system

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