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Protocol publication

[…] Sequencing data for mRNA and siRNA fractions in RNAi strains (GEO accession GSE31300) () and W303 total RNA (GEO accession GSE38383) () were quality and adapter trimmed using Trim Galore (v0.2.3; default options; http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and aligned to the yeast genome (build SGD1.01) using Bowtie () (v0.12.7; default settings plus ‘--best’) allowing non-unique sequences to be assigned at random. For expressed gene analysis (), reads overlapping each ORF were binned and only ORFs with >100 reads were used. For siRNA profiles (), reads were binned over 50 bp intervals using SeqMonk (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). For siRNA versus total RNA expression (), read counts were quantified in consecutive 100 bp bins across the genome using SeqMonk, bins with >10,0000 total RNA reads were excluded as were bins derived from 2µ sequence which is single copy in the genome sequence but high copy in reality, and a pseudocount of one read was added to total and siRNA read counts for each bin. Total RNA levels were multiplied by copy number to correct for the division of reads amongst copies that occurs during mapping, or alternate normalization was applied in (see below for the reasoning underlying this copy number normalization methodology). The copy number for each bin was determined by splitting the complete genomic sequence into overlapping 20 bp segments at 1 bp intervals and re-mapping to the genome with reads allowed to align to all perfectly matching sequences, producing a measure of the number of genomic sequences matching each 100 bp bin. Little difference was seen if one mismatch was allowed (data not shown). […]

Pipeline specifications

Software tools Trim Galore!, Bowtie, SeqMonk
Diseases Multiple Chronic Conditions