A 100% match to both the 12 bp barcode sequence and the partial NgoMIV restriction site were crucial to retain the reads from each sample.
This filter removes those duplicate reads within a 6 × 6 window with similar sequence (allowing eight mismatches), and retains the read with the highest 'chastity'chastity
Under the normal trimming scheme we trimmed the 5' and 3' ends of the reads with quality scores of lower than 20 (or 0.01 probability of error), then we retained the reads between a minimum length of 40 nt and a maximum of 85 nt with no further trimming (full filtered length).
Nevertheless, two variants that abolish splice site recognition in PTPRO in Idiopathic Nephritic Syndrome reported by Ozaltin et al. (2011) had similar phenotypes even though one retained the reading frame and the other caused a frameshift.
However, in mono-, di- and tetranucleotide repeats, most deletions result in frame-shift mutation, which are usually deleterious; only deletions of a multiple of three units retain the reading frame.
Clone D from KYM-1 cells showed both fragment lengths corresponded with an un-edited fragment length, and this was confirmed by the sequence data, which showed a single nucleotide alteration in both alleles; this mutation disrupts the FauI site, but retains the reading frame, suggesting that EZH2 remains expressed (and likely functional) in these clones.
For the de novo libraries we removed all reads showing traces of adaptor sequences, whereas for the mapping libraries we trimmed reads showing similarity to adaptor sequences and retained the read if it was longer than 30 bp.
Once this first set of alignments was produced, we also purged any overlapping reads with more than 50% overlap of the shorter read from the data set, retaining the longest read for any overlapping region.
There are 24 transcription factor gene annotations with donor or acceptor splice site selection as the sole mechanism of transcript variation, but for almost all of these the alternative splice sites are just 3, 6, 9 or 12 nucleotides apart, thereby retaining the translational reading frame.
