Genomic sequencing of single microbial cells from environmental samples

Abstract

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

Figure 1

Single cell MDA provides sufficient DNA to carry out PCR analysis of many loci. A single Borrelia cell (arrow) was isolated directly from tick midgut tissue by micromanipulation. The individual cells were amplified in 5μl MDA reactions yielding approximately 4.5μg DNA product which was used as template in PCR reactions with primers for loci previously employed for MLST analysis of cultured Borrelia isolates [49]. The PCR products were sequenced and confirmed the published genotypes of outer surface proteins (ospA, ospB, osp C). PCR using total DNA extracted from the tissue, an alternative approach, makes it impossible to determine if genotypes of different loci were genetically linked within individual cells, as the data obtained would be from all of the bacterial cells present. While culturing isolates is an alternative method that does provide linkage information, the organisms for which adequate culture conditions are available are very limited and there is often strong selection for fast growing strains biasing the results. Moreover, virulence factors associated with plasmids can be rapidly lost in culture [50].

Figure 2

Characterization of single cell MDA DNA sequence. (a) Pyrosequence depth distribution for a single amplified Flavobacterium (top panel) (TW, RS unpublished) and the single amplified TM7a (middle panel) [20] demonstrate MDA-originated high regional fluctuations in sequence coverage, while the sequence depth for the unamplified sample Xylanimonas cellulosilytica DSM 15894 exhibits a low degree of variation (bottom panel). Sequence positions were determined via arranging contigs by length. TM7a pyrosequence was screened to exclude reads contributing to contigs <300 bp and human reads. The mean sequence depth is 85.7 (± 73.7) for the flavobacterial SAG, 14.4 (± 16.2) for the TM7a SAG, and 31.9 (± 13.9) for X. cellulosilytica. A certain degree of sequence depth fluctuation may be attributed to repeats and mis-assemblies. (b) Genome coverage as function of the genome sequencing effort. Additional shotgun sequencing would not be effective in recovering the under-amplified genomic regions in the single amplified Flavobacterium (estimated genome size of ~2.3Mb) or TM7a (no genome estimate available). The X. cellulosilytica genome is estimated to be ~3.8Mb.