Dataset Information

Assessing Illumina technology for the high-throughput sequencing of bacteriophage genomes.

ABSTRACT: Bacteriophages are the most abundant biological entities on the planet, playing crucial roles in the shaping of bacterial populations. Phages have smaller genomes than their bacterial hosts, yet there are currently fewer fully sequenced phage than bacterial genomes. We assessed the suitability of Illumina technology for high-throughput sequencing and subsequent assembly of phage genomes. In silico datasets reveal that 30× coverage is sufficient to correctly assemble the complete genome of ~98.5% of known phages, with experimental data confirming that the majority of phage genomes can be assembled at 30× coverage. Furthermore, in silico data demonstrate it is possible to co-sequence multiple phages from different hosts, without introducing assembly errors.

SUBMITTER: Rihtman B

PROVIDER: S-EPMC4893331 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Assessing Illumina technology for the high-throughput sequencing of bacteriophage genomes.

Rihtman Branko B Meaden Sean S Clokie Martha R J MR Koskella Britt B Millard Andrew D AD

PeerJ 20160601

Bacteriophages are the most abundant biological entities on the planet, playing crucial roles in the shaping of bacterial populations. Phages have smaller genomes than their bacterial hosts, yet there are currently fewer fully sequenced phage than bacterial genomes. We assessed the suitability of Illumina technology for high-throughput sequencing and subsequent assembly of phage genomes. In silico datasets reveal that 30× coverage is sufficient to correctly assemble the complete genome of ~98.5% ...[more]

PMID: 27280068

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Project description:BackgroundGenetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.ResultsBased on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.ConclusionsThe high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Project description:BackgroundThe development of second generation sequencing methods has enabled large scale DNA variation studies at moderate cost. For the high throughput discovery of single nucleotide polymorphisms (SNPs) in species lacking a sequenced reference genome, we set-up an analysis pipeline based on a short read de novo sequence assembler and a program designed to identify variation within short reads. To illustrate the potential of this technique, we present the results obtained with a randomly sheared, enzymatically generated, 2-3 kbp genome fraction of six pooled Meleagris gallopavo (turkey) individuals.ResultsA total of 100 million 36 bp reads were generated, representing approximately 5-6% (approximately 62 Mbp) of the turkey genome, with an estimated sequence depth of 58. Reads consisting of bases called with less than 1% error probability were selected and assembled into contigs. Subsequently, high throughput discovery of nucleotide variation was performed using sequences with more than 90% reliability by using the assembled contigs that were 50 bp or longer as the reference sequence. We identified more than 7,500 SNPs with a high probability of representing true nucleotide variation in turkeys. Increasing the reference genome by adding publicly available turkey BAC-end sequences increased the number of SNPs to over 11,000. A comparison with the sequenced chicken genome indicated that the assembled turkey contigs were distributed uniformly across the turkey genome. Genotyping of a representative sample of 340 SNPs resulted in a SNP conversion rate of 95%. The correlation of the minor allele count (MAC) and observed minor allele frequency (MAF) for the validated SNPs was 0.69.ConclusionWe provide an efficient and cost-effective approach for the identification of thousands of high quality SNPs in species currently lacking a sequenced genome and applied this to turkey. The methodology addresses a random fraction of the genome, resulting in an even distribution of SNPs across the targeted genome.

Dataset Information

Assessing Illumina technology for the high-throughput sequencing of bacteriophage genomes.

Publications

Assessing Illumina technology for the high-throughput sequencing of bacteriophage genomes.

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