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Further, Maq's alignment scores are based on the probability of error in mismatches between read and reference (details are in [ 12]).
Specifically, sequence alignment was performed using Burrows-Wheeler Aligner (BWA) [ 41] against mouse mature miRNA sequences (downloaded from miRBase (Release 20: June 2013; http://www.mirbase.org/), while allowing one mismatch between read and reference.
However, mismatches between read and reference sequence within the barcode region (that encodes sample origin) must be partitioned from those in the target region (that would signal SNPs or mutations).
The last term, ∏ i = 1, 2 P (r n (i ) | seq m l p ) describes the probability of observed read sequences based on quality scores and base discrepancy between read and reference.
We ran TopHat in the ' paired-end mode' with the minimum distance between paired-end reads of 120 bp, a maximum distance of 500,000 bp, and default settings of other parameters (e.g., no more than two mismatches between read and reference were allowed in the first 28 bp (5' end) of the read).
Any gaps opened and extended were considered as a mismatch in calculating percent identity between read and reference.
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Subsequently, GATK28 was used to call any potential variable sites between reads and reference (HaplotypeCaller) and to ultimately generate a consensus sequence (FastaAlternateReferenceMaker)We called genotypes and generated a consensus sequence using GATK (Picard v.2.6).
As in the case of PhiX174, there were a number of small indels evident in the comparison between reads and reference which were excluded from our statistic.
After Roche/454 and/or Illumina reads are assembled, consistency between reads and reference are checked for initially indentifying candidates of SNPs and then evaluated by a Bayesian method.
This de novo assembly facilitated the mapping of reads in unique positions since it was not necessary to allow mismatches between reads and reference.
Neighboring peaks in flow value distributions, observed in earlier analyses when aligning reads to a reference, were believed to arise from biological differences between reads and reference, but by matching reads against a known mate-pair linker sequence and only using these short alignments for our analyses, we eliminate this source of error.
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