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A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes.

ABSTRACT: We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 Fundulus sp. mitochondrial genomes (192 F. heteroclitus and 191 F. majalis). Prior to sequencing, we amplified the mitochondrial genomes using 4-5 custom-made, overlapping primer pairs, and sequencing was performed on an Illumina HiSeq 2500 platform. After removing low quality and short sequences, 2.9 million and 2.8 million reads were generated for F. heteroclitus and F. majalis respectively. Individual genomes were assembled for each species by mapping barcoded reads to a reference genome. For F. majalis, the reference genome was built de novo. On average, individual consensus sequences had high coverage: 61-fold for F. heteroclitus and 57-fold for F. majalis. The approach discussed in this paper is optimized for sequencing mitochondrial genomes on an Illumina platform. However, with the proper modifications, this approach could be easily applied to other small genomes and sequencing platforms.

SUBMITTER: Nunez JC

PROVIDER: S-EPMC4978415 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes.

Nunez Joaquin C B JC Oleksiak Marjorie F MF

PloS one 20160809 8

We present a cost-effective approach to sequence whole mitochondrial genomes for hundreds of individuals. Our approach uses small reaction volumes and unmodified (non-phosphorylated) barcoded adaptors to minimize reagent costs. We demonstrate our approach by sequencing 383 Fundulus sp. mitochondrial genomes (192 F. heteroclitus and 191 F. majalis). Prior to sequencing, we amplified the mitochondrial genomes using 4-5 custom-made, overlapping primer pairs, and sequencing was performed on an Illum ...[more]

PMID: 27505419

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Project description:BackgroundNext-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools.MethodsIn this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform.ResultsTwenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25-30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2-3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample.ConclusionsThis work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples.

Dataset Information

A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes.

Publications

A Cost-Effective Approach to Sequence Hundreds of Complete Mitochondrial Genomes.

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