Manual examination of alignments showed that such indels occur not only as extensions to pre-existing structures, but are introduced in existing domains into the middle of the structure.
Sequences with 10% or more undetermined bases or chimeric features, as determined by screening with MALLARD [10] followed by manual examination of alignments generated by BLAST against GenBank, were removed.
Examination of alignments of the full-length proteins revealed 10 fully conserved residues (R112, G129, N131, R147, G151, E153, N164, Y166, G211, D213 where the numbers refer to the alignment positions in Fig. S2); all were located within the β-domain.
The examination of alignments of reads in the clusters for potential alternative splicing identified different splice variants in ca. 9% of the contigs containing reads from more than 1 cDNA insert, or ca. 3% of all genes examined.
These 10 protein coding gene regions were aligned by eye to the dataset used in Near et al. [ 40] and confirmed by examination of alignments of the inferred amino acid sequences.
Closer examination of alignments using reference sequences from the IMGT/HLA database and the IGV browser showed that the MIT-NGS calls were correct rather than the original SBT results.
Examination of the alignment found two sub-populations of sequences that did not fit the overall alignment consensus in these regions (Table S2).
Although this may seem inconsistent, further examination of the alignment of probes to gene revealed that different probesets for Tsn aligned to different populations of alternatively polyadenylated transcripts.
Alignments performed using Bowtie 2 resulted in an even greater number of miRNAs with higher counts; examination of the alignment output revealed that many of these were attributed to the allowance of insertions and deletions.
