Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology

Abstract

BackgroundStructural variants (SVs) are less common than single nucleotide polymorphisms and indels in the population, but collectively account for a significant fraction of genetic polymorphism and diseases. Base pair differences arising from SVs are on a much higher order (>100 fold) than point mutations; however, none of the current detection methods are comprehensive, and currently available methodologies are incapable of providing sufficient resolution and unambiguous information across complex regions in the human genome. To address these challenges, we applied a high-throughput, cost-effective genome mapping technology to comprehensively discover genome-wide SVs and characterize complex regions of the YH genome using long single molecules (>150 kb) in a global fashion.ResultsUtilizing nanochannel-based genome mapping technology, we obtained 708 insertions/deletions and 17 inversions larger than 1 kb. Excluding the 59 SVs (54 insertions/deletions, 5 inversions) that overlap with N-base gaps in the reference assembly hg19, 666 non-gap SVs remained, and 396 of them (60%) were verified by paired-end data from whole-genome sequencing-based re-sequencing or de novo assembly sequence from fosmid data. Of the remaining 270 SVs, 260 are insertions and 213 overlap known SVs in the Database of Genomic Variants. Overall, 609 out of 666 (90%) variants were supported by experimental orthogonal methods or historical evidence in public databases. At the same time, genome mapping also provides valuable information for complex regions with haplotypes in a straightforward fashion. In addition, with long single-molecule labeling patterns, exogenous viral sequences were mapped on a whole-genome scale, and sample heterogeneity was analyzed at a new level.ConclusionOur study highlights genome mapping technology as a comprehensive and cost-effective method for detecting structural variation and studying complex regions in the human genome, as well as deciphering viral integration into the host genome.Electronic supplementary materialThe online version of this article (doi:10.1186/2047-217X-3-34) contains supplementary material, which is available to authorized users.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length

“…15 The labeled DNA has fluorescent dUTP inserted at the Nt.BspQI nicking site and YOYO-1 (Invitrogen) in the backbone at a ratio of 40 bp per dye molecule. Image data were collected on a custom-built epifluorescence microscope consisting of an Olympus IX-71 microscope body with an Olympus 60x air NA 0.9 objective.…”

“…These massive stretches of labeled DNA greatly facilitate the assembly of genomic maps for anchoring sequencing data, 2 and they are especially important for the analysis of structural variations. 3,15 Understanding the probability distribution governing the polymer extension between labeled points along nanochannelconfined DNA is critical to accurately map the physical distance X (in nm) between probes to their genomic distance, L (in base pairs (bp)). However, the theory surrounding DNA confinement for channel sizes D ≈ l p is contentious.…”

Improved maize reference genome with single-molecule technologies

“…A structural variant was identified as an alignment outlier 32,49 , defined as two well aligned regions separated by a poorly aligned region with a large size difference between the reference genome and the map or by one or more unaligned sites, or alternatively as a gap between two local alignments. A confidence score was generated by comparing the non normalized P values of the two well aligned regions and the non normalized log likelihood ratio 50 of the unaligned or poorly aligned region. With a confidence score threshold of 3, RefSplit is sensitive to insertions and deletions as small as 100 bp (events smaller than 1 kb are generally compound or substitution and include label changes, not just spacing differences) and other changes such as inversions and complex events which could be balanced.…”

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

“…The size distribution was lower than expected and is probably a result of impurities during high-molecular-weight gDNA isolation that would cause shearing and inhibition of enzymes. Molecules were de novo assembled as previously described 32 . Two genome maps were assembled at different stringencies, map set 1 has 402 maps with an N50 length of 725 kb and spans 216 Mb (Extended Data Fig.…”