One by one: Single molecule tools for genomics

Abstract

Much of the effort in any genomics programme arises from the need to generate and purify large numbers of identical molecules, since most analytical tools rely on the analysis of bulk DNA. Biological steps such as bacterial cloning--commonly used to prepare bulk samples of defined DNA fragments--are capricious and introduce their own restrictions and distortions. The analysis of single molecules, either directly or by in vitro enzymatic amplification, makes possible the examination of native genomic DNA without the complications and restrictions of biological propagation. Techniques already exist for the in vitro propagation of genomic fragments and for genome mapping, and offer the advantages of speed, flexibility and predictable behaviour. Single molecule sequencing, for which many approaches are being developed, is more challenging, but offers even greater rewards in terms of throughput and read length.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma

“…1. In this study, we used the sequencing-by-synthesis Solexa method (18). As the maternal plasma DNA (maternal and fetal) molecules were already fragmented in nature (19), no further fragmentation was required.…”

“…We previously (15) proposed that the recently available massively parallel genomic sequencing (MPGS) platforms (16,17) might be adaptable as an approach to quantify DNA sequences for the noninvasive prenatal diagnosis of fetal chromosomal aneuploidy. In this study, we demonstrate the use of the ''Solexa'' sequencing technique (Illumina) (18) for this purpose.…”

Direct, genome-wide assessment of DNA mutations in single cells

“…In principle, such low abundant mutations can be detected by single-molecule sequencing (25). However, this necessarily involves the sequencing of fragments derived from different cells and precludes insight into the mosaic basis of somatic mutations in tissues or cell populations.…”

Somatic mutations, genome mosaicism, cancer and aging

“…However, for point mutations such artifacts are unlikely. An error introduced early in MDA would be found in only 12.5% of the sequencing reads at a given locus in a diploid cell on average [39]. Since as shown in Figure 3 true somatic mutations should affect one allele and therefore need to be found in 50% of the reads, amplification errors are easily filtered out.…”