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Open Access Highly Accessed Research article

Using paired-end sequences to optimise parameters for alignment of sequence reads against related genomes

Abhirami Ratnakumar12, Sean McWilliam1, Wesley Barris1 and Brian P Dalrymple1*

Author Affiliations

1 CSIRO Livestock Industries, 306 Carmody Road, St. Lucia, QLD 4067, Australia

2 Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 751 23 Uppsala, Sweden

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BMC Genomics 2010, 11:458  doi:10.1186/1471-2164-11-458

Published: 3 August 2010

Abstract

Background

The advent of cheap high through-put sequencing methods has facilitated low coverage skims of a large number of organisms. To maximise the utility of the sequences, assembly into contigs and then ordering of those contigs is required. Whilst sequences can be assembled into contigs de novo, using assembled genomes of closely related organisms as a framework can considerably aid the process. However, the preferred search programs and parameters that will optimise the sensitivity and specificity of the alignments between the sequence reads and the framework genome(s) are not necessarily obvious. Here we demonstrate a process that uses paired-end sequence reads to choose an optimal program and alignment parameters.

Results

Unlike two single fragment reads, in paired-end sequence reads, such as BAC-end sequences, the two sequences in the pair have a known positional relationship in the original genome. This provides an additional level of confidence over match scores and e-values in the accuracy of the positional assignment of the reads in the comparative genome. Three commonly used sequence alignment programs: MegaBLAST, Blastz and PatternHunter were used to align a set of ovine BAC-end sequences against the equine genome assembly. A range of different search parameters, with a particular focus on contiguous and discontiguous seeds, were used for each program. The number of reads with a hit and the number of read pairs with hits for the two end sequences in the tail-to-tail paired-end configuration were plotted relative to the theoretical maximum expected curve. Of the programs tested, MegaBLAST with short contiguous seed lengths (word size 8-11) performed best in this particular task. In addition the data also provides estimates of the false positive and false negative rates, which can be used to determine the appropriate values of additional parameters, such as score cut-off, to balance sensitivity and specificity. To determine whether the approach also worked for the alignment of shorter reads, the first 240 bases of each BAC end sequence were also aligned to the equine genome. Again, contiguous MegaBLAST performed the best in optimising the sensitivity and specificity with which sheep BAC end reads map to the equine and bovine genomes.

Conclusions

Paired-end reads, such as BAC-end sequences, provide an efficient mechanism to optimise sequence alignment parameters, for example for comparative genome assemblies, by providing an objective standard to evaluate performance.