RT Journal Article
SR Electronic
T1 Conversion of Genomic DNA to Proxy Constructs Suitable for Accurate Nanopore Sequencing
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 027284
DO 10.1101/027284
A1 Dimitra Tsavachidou
YR 2016
UL http://biorxiv.org/content/early/2016/02/06/027284.abstract
AB Nanopore sequencing at single-base resolution is challenging. There are developing technologies to convert DNA molecules to expanded constructs. Such constructs can be sequenced by nanopores in place of the original DNA molecules. We present a novel method for converting genomic DNA to expanded constructs (“proxies”) with 99.67% accuracy. Our method “reads” each base in each DNA fragment and appends an oligonucleotide to the DNA fragment after each base “reading”. Each appended oligonucleotide represents a specific base type, so that the proxy construct consisting of all the appended oligonucleotides faithfully represents the original DNA sequence. We generated proxies for genomic DNA and confirmed the identities of both the proxies and their corresponding original DNA sequences by performing sequencing using Ion Torrent sequencer.Conversion to proxies had only 0.33% raw error rate. Errors were: 93.96% deletions, 5.29% insertions, and 0.74% substitutions. The longest sequenced proxy was 170 bases, corresponding to a 17-base original DNA sequence. The short length of the detected proxies reflected restrictions imposed by Ion Torrent’s short reads and was not caused by limitations of our method. The consensus sequence built by using proxies alone (average length: 120 bases; corresponding to original sequences with average length 12 bases) covered 55% of the reference genome with 100% accuracy, and outperformed the Ion Torrent sequencing of the corresponding original DNA fragments in terms of accuracy, coverage and number of aligned sequences. Data and other materials can be found at http://www.vastogen.com/data.html. This proof-of-concept experiment demonstrates highly accurate proxy construction at the whole genome level. To our knowledge, this is the first demonstrated construction of expanded versions of DNA at the whole genome level.