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While some fragmented sequences are removed, which helps with alignment performance by limiting the number of gaps in the MSA [4], the majority of the removed sequences are simply highly similar (greater than 95%) and therefore redundant.
Besides differences in statistical power, another factor that may explain the difference between the 2010 and the present study is that the six removed sequences are recombinants, and it is known that recombination interferes with likelihood methods for detecting selection pressure on codon alignments (Anisimova et al. 2003).
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Briefly, low quality (removed, sequences were aligned against an orangutan (P. abelii) genome (3 ), and nonmapped sequences were subjected to de novo assembly.
We then further removed sequences that were read only once and 6,870,535 reads remained.
We also discarded duplicate reads from our data (duplicates can be removed if sequences are exactly the same, without allowing any mismatches, because they are considered PCR duplicates).
After redundancy was removed, sequences ≥ 18 nt were mapped to the human genome (UCSC hg18) using SOAP.
A total of 5.2 × 107 bases from 15,560 cleaned (redundancy removed) sequence documents were analyzed.
These were then filtered to remove sequences that were unlikely to represent U1/U2 introns (see Materials and Methods).
In step 4, the user supplies a length error cutoff value to remove sequences that are excessively short or long.
MUSCLE alignments were inspected visually to remove sequences that were obvious alignment errors.
Adaptors were removed, and sequences were trimmed for length and quality with standard settings.
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