Softgenetics has added a function to its Nextgene software that provides Sanger-quality sequence from short reads from next-generation sequencing platforms such as the Illumina genome analyser (GA).
Short-read lengths produced by next-generation sequencers such as the Illumina GA can create difficulty for accurate data analysis.
Relatively high error rates (compared to other technologies such as Sanger sequencing) further complicate the analysis of next-generation sequencing data.
For these reasons, lengthening the short reads prior to analysis and statistically correcting or removing errors help to improve alignment accuracy, Indel detection and assembly.
An accurate method to elongate reads, utilising paired-end reads, has been developed by Softgenetics for its Nextgene software.
Megan Manion, Nextgene product specialist, said: 'Sequencing paired-end reads is a useful technique that produces reads in pairs such that each pair of reads are a known distance from each other in the genome.
'This is accomplished by preparing DNA fragments of a certain length [200bp, for example].
'This fragment size, or library size, is the distance between each pair of reads.
'Sequencing is then done from each end of the fragment, producing two paired reads.
'Nextgene's paired-end merging technique takes advantage of paired-end information, along with the additional coverage from sequenced overlapping DNA fragments, to produce long reads spanning the entire library size with an extremely low error rate.
'Paired-end reads can be merged by elongating the paired reads to the point that there is overlap between the two reads.
'This allows the paired reads to be joined together to form one continuous, longer read.
'The number of elongation cycles required depends on the read lengths and the library size.
'Each cycle of condensation will generally increase the average read length to 1.6 the original length for shorter [<=36 bp] reads and to six bases less than twice the original length for longer [>36bp] reads.
'These values may be reduced with an average depth of coverage less than 30x.
'A single cycle of elongation of 75bp reads from a 200bp library, for example, allows the paired reads to overlap and be linked together.
'For 35bp reads from a 200bp library, three cycles of elongation allow for the linking of the paired reads.
'Reads should be extended until a significant portion of the paired reads [roughly 15 per cent of the elongated read length] will be expected to overlap,' she added.
