library construction induced mutation rate from expressed sequence tag collections

Estimation of population heterozygosity and library construction induced mutation rate from expressed sequence tag collections. Abstract: Unigene alignments obtained from cDNA libraries made using multiple individuals are not currently used to estimate population heterozygosity, as they are known to harbor mutations created during library construction. We describe an estimator of population heterozygosity that only utilizes SNPs unlikely to be library construction artifacts.-3-Expressed Sequence Tag (EST) projects have become a popular and cost-effective means of initially cataloging a large number of genes in biological systems without genome projects (reviewed in Rudd, 2003). DNA sequencing of several thousand randomly chosen clones from a cDNA library allows thousands of different transcripts to be identified. However, since the likelihood of observing a given transcript is proportional to the expression level of that transcript in the tissue from which the library is derived, oftentimes transcripts are represented by several EST sequences. In a typical EST project, using an inbred line as the starting material to construct the cDNA library, ESTs associated with the same transcript can be assembled into a Unigene cluster and the consensus sequence associated with that assembly is referred to as a Unigene. On the other hand, if the ESTs contributing to a Unigene cluster are associated with cDNA libraries obtained from different individuals, or different inbred lines, then Single Nucleotide Polymorphisms (SNPs) can be identified from the resulting alignments (Picoult-Newberg et al., 1999). Although SNPs obtained from haphazard collections of ESTs may have utility as markers, it would be difficult to estimate per site heterozygosity from such a resource, since the unknown ascertainment scheme could bias any estimates. On the other hand, if a cDNA library were constructed from an equimolar collection of RNAs from an infinite number of outbred individuals, the alignments associated with different Unigene clusters could be used to estimate per site heterozygosity using standard population genetics methods for estimating diversity (e.g., Hartl and Clark, 1997). Standard methods could also be applied to a Unigene cluster obtained from a library derived from a finite number of individuals provided the alignment depth of that cluster is much less than twice the number of individuals used to-4-create the cDNA library, in order to insure alleles sampled in the Unigene cluster are likely to be independent of one another. However, the application of standard methods for estimating per site heterozygosity to collections of ESTs has generally been avoided, as the DNA …