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Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

ABSTRACT: Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1-3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a 'boundary switch') or the conversion of a repeat within a block to a different repeat type ('modular structure' mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood.

SUBMITTER: Shanks ME

PROVIDER: S-EPMC2599865 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Shanks Morag E ME May Celia A CA Dubrova Yuri E YE Balaresque Patricia P Rosser Zoë H ZH Adams Susan M SM Jobling Mark A MA

Mutation research 20080925 1-2

Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 ...[more]

PMID: 18929582

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Project description:The advent of Next Generation Sequencing (NGS) has led to the generation of enormous volumes of short read sequence data, cheaply and in reasonable time scales. Nevertheless, the quality of genome assemblies generated using NGS technologies has been greatly affected, compared to those generated using Sanger DNA sequencing. This is largely due to the inability of short read sequence data to scaffold repetitive structures, creating gaps, inversions and rearrangements and resulting in assemblies that are, at best, draft forms. Third generation single-molecule sequencing (SMS) technologies (e.g. Pacific Biosciences Single Molecule Real Time (SMRT) system) address this challenge by generating sequences with increased read lengths, offering the prospect to better recover these complex repetitive structures, concomitantly improving assembly quality.Here, we evaluate the ability of SMS data (specifically human genome Pacific Biosciences SMRT data) to recover poorly represented repetitive sequences (specifically, GC-rich human minisatellites). To do this we designed a pipeline for the collection, processing and local assembly of single-molecule sequence data to form accurate contiguous local reconstructions. Our results show the recovery of an allele of the non-coding minisatellite MS1 (located on chromosome 1 at 1p33-35) at greater than 97% identity to reference (GRCh38) from the unprocessed sequence data of a haploid complete hydatidiform mole (CHM1) cell line. Furthermore, our assembly revealed an allele of over 500 repeat units; much larger than the reference (GRCh38), but consistent in structure with naturally occurring alleles that are segregating in human populations. This local assembly's reconstruction was validated with the release of the whole genome assemblies GCA_001297185.1 and GCA_000772585.3, where this allele occurs. Additionally, application of this pipeline to coding minisatellites in the PRDM9 and ZNF93 genes enabled recovery of high identity allele structures for these sequence regions whose length was confirmed by PCR from cell line genomic DNA. The internal repeat structure of the PRDM9 allele recovered was consistent with common human-specific alleles.Code available at https://github.com/ndliberial/smrt_pipeline.dno2@le.ac.uk.

Dataset Information

Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Publications

Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

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