NGS method, RNA seq

Method in Lei 2013, Gene, Diminishing returns in next-generation sequencing (NGS)

transcriptome data. 

The sequencing files downloaded from NCBI SRA database were initially

converted from SRA format to FASTQ format using SRA toolkit

(http://www.ncbi.nlm.nih.gov/Traces/sra/?view=software). Then, the

raw data were filtered using the following criteria: (1) the number of

unknown bases (N) was no more than two for each read; and (2) the

fraction of low quality sites (Q b 5) was no more than 50% for each

read. The data that passed this quality control were then used to map

back to their respective genome sequences using bowtie2 (Langmead

and Salzberg, 2012). Only uniquely mapped reads with no more than

two mismatches were retained for further analysis. After mapping, the

counts for each gene were summarized using HTSeq (http://wwwhuber.

embl.de/users/anders/HTSeq/doc/overview.html). In the simulation,

a predetermined-sized subset of reads was randomly selected

from the original file. Using the samemapping procedure as mentioned

above, the RPKM for each gene and depth of coverage were calculated

and comparedwith those fromoriginal data. In-house Perl and R scripts

were developed for data analysis and graphing (available upon request).