Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Transcriptome bioinformatic analysis.

JF Jeffrey A. Freiberg
YB Yoann Le Breton
BT Bao Q. Tran
AS Alison J. Scott
JH Janette M. Harro
RE Robert K. Ernst
YG Young Ah Goo
EM Emmanuel F. Mongodin
DG David R. Goodlett
KM Kevin S. McIver
MS Mark E. Shirtliff
request Request a Protocol
ask Ask a question
Favorite

RNA sequencing datasets in FastQ format were analyzed for quality using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (84). Reads were trimmed and Illumina adapters were clipped using Trimmomatic v 0.32 (85) with a leading and trailing minimum score of 3 and a 4-base sliding window minimum score of 15, which resulted in an average of 99.98% of reads surviving (range, 99.93% to 99.99%). Reads were mapped to the GAS MGAS5005 genome (NC_007297.1; NCBI) using Bowtie2 v 2.2.4 (86) run in end-to-end mode with default settings for an average overall alignment rate of 98.80% (range, 95.72% to 99.38%). Transcript abundances were calculated in fragments per kilobase per million mapped reads (FPKM) using Cufflinks v 2.2.1 (87) with a ribosomal masking file for all 5S, 16S, 23S, and tRNA loci (NC_007297.1.gff; NCBI). Cuffdiff (88), a program within the Cufflinks package, was used to calculate differential expression values for genes with an FDR-adjusted P value (q value) of less than 0.01. Operon structure was predicted from the resulting Bowtie2 alignment files using Rockhopper v 2.03 (89, 90).

Copyright and License information:  ©2016 Freiberg et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A