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Whole genome amplification and de novo assembly of single bacterial cells.

ABSTRACT: Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples.

SUBMITTER: Rodrigue S

PROVIDER: S-EPMC2731171 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Whole genome amplification and de novo assembly of single bacterial cells.

Rodrigue Sébastien S Malmstrom Rex R RR Berlin Aaron M AM Birren Bruce W BW Henn Matthew R MR Chisholm Sallie W SW

PloS one 20090902 9

<h4>Background</h4>Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.<h4>Methodology/principal findings</h4>We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while ...[more]

PMID: 19724646

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Project description:BackgroundNext-generation sequencing (NGS) technology has paved the way for rapid and cost-efficient de novo sequencing of bacterial genomes. In particular, the introduction of PCR-free library preparation procedures (LPPs) lead to major improvements as PCR bias is largely reduced. However, in order to facilitate the assembly of Illumina paired-end sequence data and to enhance assembly performance, an increase of insert sizes to facilitate the repeat bridging and resolution capabilities of current state of the art assembly tools is needed. In addition, information concerning the relationships between genomic GC content, library insert size and sequencing quality as well as the influence of library insert size, read length and sequencing depth on assembly performance would be helpful to specifically target sequencing projects.ResultsOptimized DNA fragmentation settings and fine-tuned resuspension buffer to bead buffer ratios during fragment size selection were integrated in the Illumina TruSeq(®) DNA PCR-free LPP in order to produce sequencing libraries varying in average insert size for bacterial genomes within a range of 35.4-73.0 % GC content. The modified protocol consumes only half of the reagents per sample, thus doubling the number of preparations possible with a kit. Examination of different libraries revealed that sequencing quality decreases with increased genomic GC content and with larger insert sizes. The estimation of assembly performance using assembly metrics like corrected NG50 and NGA50 showed that libraries with larger insert sizes can result in substantial assembly improvements as long as appropriate assembly tools are chosen. However, such improvements seem to be limited to genomes with a low to medium GC content. A positive trend between read length and assembly performance was observed while sequencing depth is less important, provided a minimum coverage is reached.ConclusionsBased on the optimized protocol developed, sequencing libraries with flexible insert sizes and lower reagent costs can be generated. Furthermore, increased knowledge about the interplay of sequencing quality, insert size, genomic GC content, read length, sequencing depth and the assembler used will help molecular biologists to set up an optimal experimental and analytical framework with respect to Illumina next-generation sequencing of bacterial genomes.

Dataset Information

Whole genome amplification and de novo assembly of single bacterial cells.

Publications

Whole genome amplification and de novo assembly of single bacterial cells.

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