Dataset Information

Illumina sequencing of 15 deafness genes using fragmented amplicons.

ABSTRACT: BACKGROUND: Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology. METHODS: We performed Illumina sequencing in 4 patients on 563 multiplexed amplicons covering the exons of 15 genes involved in the hearing process. After exploring several fragmentation strategies, the amplicons were fragmented using Covaris sonication prior to library preparation. CLC genomic workbench was used to analyze the data. RESULTS: We achieve an excellent coverage with more than 99% of the amplicons bases covered. All variants that were previously validated using Sanger sequencing, were also called in this study. Variant calling revealed less false positive and false negative results compared to the previous study. For each patient, several variants were found that are reported by ClinVar as possible hearing loss variants. CONCLUSION: Migration from GS FLX amplicon sequencing to Illumina amplicon sequencing is straightforward and leads to more accurate results.

SUBMITTER: Van Nieuwerburgh F

PROVIDER: S-EPMC4266979 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Illumina sequencing of 15 deafness genes using fragmented amplicons.

Van Nieuwerburgh Filip F De Keulenaer Sarah S De Schrijver Joachim J Vandesompele Jo J Van Criekinge Wim W Coucke Paul J PJ Deforce Dieter D

BMC research notes 20140809

<h4>Background</h4>Resequencing of deafness related genes using GS FLX massive parallel sequencing of PCR amplicons spanning selected genes has previously been reported as a successful strategy to discover causal variants. The amplicon lengths were designed to be smaller than the sequencing read length of GS FLX technology, but are longer than Illumina sequencing technology read lengths. Fragmentation is thus required to sequence these amplicons using high throughput Illumina technology.<h4>Meth ...[more]

PMID: 25106482

Similar Datasets

Project description:In this paper we explore high-throughput Illumina sequencing of nuclear protein-coding, ribosomal, and mitochondrial genes in small, dried insects stored in natural history collections. We sequenced one tenebrionid beetle and 12 carabid beetles ranging in size from 3.7 to 9.7 mm in length that have been stored in various museums for 4 to 84 years. Although we chose a number of old, small specimens for which we expected low sequence recovery, we successfully recovered at least some low-copy nuclear protein-coding genes from all specimens. For example, in one 56-year-old beetle, 4.4 mm in length, our de novo assembly recovered about 63% of approximately 41,900 nucleotides in a target suite of 67 nuclear protein-coding gene fragments, and 70% using a reference-based assembly. Even in the least successfully sequenced carabid specimen, reference-based assembly yielded fragments that were at least 50% of the target length for 34 of 67 nuclear protein-coding gene fragments. Exploration of alternative references for reference-based assembly revealed few signs of bias created by the reference. For all specimens we recovered almost complete copies of ribosomal and mitochondrial genes. We verified the general accuracy of the sequences through comparisons with sequences obtained from PCR and Sanger sequencing, including of conspecific, fresh specimens, and through phylogenetic analysis that tested the placement of sequences in predicted regions. A few possible inaccuracies in the sequences were detected, but these rarely affected the phylogenetic placement of the samples. Although our sample sizes are low, an exploratory regression study suggests that the dominant factor in predicting success at recovering nuclear protein-coding genes is a high number of Illumina reads, with success at PCR of COI and killing by immersion in ethanol being secondary factors; in analyses of only high-read samples, the primary significant explanatory variable was body length, with small beetles being more successfully sequenced.

Dataset Information

Illumina sequencing of 15 deafness genes using fragmented amplicons.

Publications

Illumina sequencing of 15 deafness genes using fragmented amplicons.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets