RNA-seq Pipeline for Known Transcripts - Revision history

Davebrid: added section for aligning reads with bowtie

2011-11-08T16:08:32Z

added section for aligning reads with bowtie

← Older revision		Revision as of 16:08, 8 November 2011
Line 27:		Line 27:
	</pre>		</pre>
	# Filter for quality, if applicable		# Filter for quality, if applicable
	# Trim, if applicable		# Trim, if applicable using Fast-x. The following keeps 100% of the reads with a quality of 25 or greater:
			<pre>
			fastq_quality_filter -v -q 25 -p 100 -i control-reads.txt -o control-reads-quality25.txt
			</pre>

	==~~Generate a Reference Genome~~==		==Align Reads==
	~~# Run bowtie-build to generate Burroughs Wheeler transformed reference genome (.ebwt format)~~.		This can be done with either TopHat or Bowtie, so choose one of the following. The reference genomes are located in the following locations:
	~~# http:~~//~~bowtie~~-~~bio.sourceforge.net~~/~~index.shtml (bowtie, tophat, and cufflinks are here).~~		<pre>
	~~# [Optional input and parameter settings are in square brackets.]~~		/database/davebrid/RNAseq/reference-genomes/hg19
	# <~~Required parameters are in greater than~~/~~less than brackets.~~>		/database/davebrid/RNAseq/reference-genomes/mm9
	~~# This BW transformed~~ reference ~~genome can be created once then used repeatedly in the future~~.		</pre>
	~~# $ is the command prompt~~.		These reference alignments are pre-built UCSC genomes and downloaded from ftp://ftp.cbcb.umd.edu/pub/data/bowtie_indexes/

			===Align Reads to Reference Genome with Bowtie===
			Run bowtie to align reads to reference genomes. The following generates a sam formatted alignment using the best quality flag for reads aligned to hg19
	<pre>		<pre>
	$ bowtie-~~build [~~-~~f specifies reference genome is in fasta format] <path to input reference genome (e.g.~~ /~~ccmb~~/~~CoreBA~~/~~BioinfCore~~/~~Common~~/~~DATA/BowtieData/H_Sapiens~~/hg19.~~fa)> <base name for reference genome output .ebwt files (e~~.~~g hg19)>~~		bowtie --sam --best /database/davebrid/RNAseq/reference-genomes/hg19/hg19 control-reads-quality25.txt control-aligned-quality25.sam
	</pre>		</pre>
	==Align Reads to Reference Genome with Tophat==
			===Align Reads to Reference Genome with Tophat===
	Run tophat to align reads to the reference genome. I’ve included a pseudo command line as well as a “real” command line.		Run tophat to align reads to the reference genome. I’ve included a pseudo command line as well as a “real” command line.
	<pre>		<pre>

Davebrid: added seqanswers link

2011-11-08T16:02:47Z

added seqanswers link

← Older revision		Revision as of 16:02, 8 November 2011
Line 1:		Line 1:
			==Reference Pages==
			* http://seqanswers.com/wiki/How-to/RNASeq_analysis

	==Sequence Quality and Trimming==		==Sequence Quality and Trimming==
	# Run FASTQC to assess quality of reads from sequencer and:		# Run FASTQC to assess quality of reads from sequencer and:

Davebrid: removed linebreak

2011-10-21T13:31:01Z

removed linebreak

← Older revision		Revision as of 13:31, 21 October 2011
Line 21:		Line 21:
	* Example command where s_2_100.txt is the original file, mybarcodes.txt is the barcode file, 2 mismatches are allowed (default is 1). This will generate files /tmp/bla_BC#.txt:		* Example command where s_2_100.txt is the original file, mybarcodes.txt is the barcode file, 2 mismatches are allowed (default is 1). This will generate files /tmp/bla_BC#.txt:
	<pre>		<pre>
	cat s_2_100.txt \| /usr/local/bin/fastx_barcode_splitter.pl --bcfile mybarcodes.txt --bol --mismatches 2 \		cat s_2_100.txt \| /usr/local/bin/fastx_barcode_splitter.pl --bcfile mybarcodes.txt --bol --mismatches 2 --prefix /tmp/bla_ --suffix ".txt"
	--prefix /tmp/bla_ --suffix ".txt"
	</pre>		</pre>
	# Filter for quality, if applicable		# Filter for quality, if applicable

Davebrid: added instructions for barcode splitting

2011-10-21T13:30:27Z

added instructions for barcode splitting

@@ Line 6: / Line 6: @@
 ===Filter Sequences Using FastX-Toolkit===
 # Filter for quality, if applicable
 # Trim, if applicable

Davebrid: added link to FastQC report analysis

2011-10-03T20:13:13Z

added link to FastQC report analysis

← Older revision		Revision as of 20:13, 3 October 2011
Line 3:		Line 3:
	# FASTQC available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/		# FASTQC available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
	## Open run_fastqc on a windows machine. Individually open each sequence file and allow it to analyse. Save this report.		## Open run_fastqc on a windows machine. Individually open each sequence file and allow it to analyse. Save this report.
	## Check this report to decide if sequences need to be trimmed or discarded.		## Check this report to decide if sequences need to be trimmed or discarded. See http://bioinfo.cipf.es/courses/mda11/lib/exe/fetch.php?media=ngs_qc_tutorial_mda_val_2011.pdf for sample results and an explanation of the quality report

	===Filter Sequences Using FastX-Toolkit===		===Filter Sequences Using FastX-Toolkit===

Davebrid: added information about using FastQC

2011-10-03T20:12:14Z

added information about using FastQC

← Older revision		Revision as of 20:12, 3 October 2011
Line 1:		Line 1:
	==Sequence Quality and Trimming==		==Sequence Quality and Trimming==
	# Run FASTQC to assess quality of reads from sequencer and:		# Run FASTQC to assess quality of reads from sequencer and:
			# FASTQC available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/
			## Open run_fastqc on a windows machine. Individually open each sequence file and allow it to analyse. Save this report.
			## Check this report to decide if sequences need to be trimmed or discarded.

			===Filter Sequences Using FastX-Toolkit===
	# Filter for quality, if applicable		# Filter for quality, if applicable
	# Trim, if applicable		# Trim, if applicable
	~~# FASTQC available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/~~

	==Generate a Reference Genome==		==Generate a Reference Genome==

Davebrid: added categories

2011-09-14T14:00:31Z

added categories

← Older revision		Revision as of 14:00, 14 September 2011
Line 37:		Line 37:
	$ cuffdiff -o ./HG19/Cuffdiff_out_options_b_u_N_compatible/ -p 14 -L Control,PUF_kd --no-update-check -b /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa -u -N --compatible-hits-norm /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/cuffcompare_out.combined.gtf /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_001_trimmed/accepted_hits.bam /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_002_trimmed/accepted_hits.bam		$ cuffdiff -o ./HG19/Cuffdiff_out_options_b_u_N_compatible/ -p 14 -L Control,PUF_kd --no-update-check -b /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa -u -N --compatible-hits-norm /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/cuffcompare_out.combined.gtf /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_001_trimmed/accepted_hits.bam /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_002_trimmed/accepted_hits.bam
	</pre>		</pre>
			[[Category: Bioinformatics]]
			[[Category: Transcription]]
			[[Category: RNA-seq]]
			[[Category: Next Generation Sequencing]]

Davebrid: updated formatting

2011-09-14T13:37:15Z

updated formatting

← Older revision		Revision as of 13:37, 14 September 2011
Line 1:		Line 1:
	~~RNA-Seq pipeline – known transcripts~~		==Sequence Quality and Trimming==
	1. Run ~~FASTQ~~ to assess quality of reads from sequencer and:		# Run FASTQC to assess quality of reads from sequencer and:
	a. Filter for quality, if applicable		# Filter for quality, if applicable
	b. Trim, if applicable		# Trim, if applicable
	c. http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/		# FASTQC available at http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

	2. Run bowtie-build to generate Burroughs Wheeler transformed reference genome (.ebwt format).		==Generate a Reference Genome==
	a. http://bowtie-bio.sourceforge.net/index.shtml (bowtie, tophat, and cufflinks are here).		# Run bowtie-build to generate Burroughs Wheeler transformed reference genome (.ebwt format).
	b. [Optional input and parameter settings are in square brackets.]		# http://bowtie-bio.sourceforge.net/index.shtml (bowtie, tophat, and cufflinks are here).
	c. <Required parameters are in greater than/less than brackets.>		# [Optional input and parameter settings are in square brackets.]
	d. This BW transformed reference genome can be created once then used repeatedly in the future.		# <Required parameters are in greater than/less than brackets.>
	e. $ is the command prompt.		# This BW transformed reference genome can be created once then used repeatedly in the future.
			# $ is the command prompt.

			<pre>
	$ bowtie-build [-f specifies reference genome is in fasta format] <path to input reference genome (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa)> <base name for reference genome output .ebwt files (e.g hg19)>		$ bowtie-build [-f specifies reference genome is in fasta format] <path to input reference genome (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa)> <base name for reference genome output .ebwt files (e.g hg19)>
			</pre>
	3. Run tophat to align reads to the reference genome. I’ve included a pseudo command line as well as a “real” command line.		==Align Reads to Reference Genome with Tophat==
			Run tophat to align reads to the reference genome. I’ve included a pseudo command line as well as a “real” command line.
			<pre>
	$ tophat [-p #processors -o ./output_directory] <./reference genome in both .ebwt and fasta formats (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19)> <reads file to be aligned (e.g. s_1_1_sequence.fastq)>		$ tophat [-p #processors -o ./output_directory] <./reference genome in both .ebwt and fasta formats (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19)> <reads file to be aligned (e.g. s_1_1_sequence.fastq)>
	$ tophat -p 5 -o ./HG19/tophat_out_hg19_001_trimmed /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19 ./HG19/Rich_trim/A_1_16_85.fastq		$ tophat -p 5 -o ./HG19/tophat_out_hg19_001_trimmed /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19 ./HG19/Rich_trim/A_1_16_85.fastq
			</pre>
	4. Run cuffcompare to create .gtf format reference genome from a generic reference genome. Note that cuffcompare adds the tss_id and p_id columns that you will need in cuffdiff. This .gtf reference can be created once then used repeatedly in the future.		==Use Cuffcompare to Generate .gtf Reference==
			Run cuffcompare to create .gtf format reference genome from a generic reference genome. Note that cuffcompare adds the tss_id and p_id columns that you will need in cuffdiff. This .gtf reference can be created once then used repeatedly in the future.
			<pre>
	$ cuffcompare [-o ./output_directory] < input file twice (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf )>		$ cuffcompare [-o ./output_directory] < input file twice (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf )>

	$ cuffcompare -o ./cuffcompare_out /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf		$ cuffcompare -o ./cuffcompare_out /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf
			</pre>

			==Use Cuffdiff to Identify Differentially Expressed Transcripts==
	5. Run cuffdiff to identify differentially abundant transcripts.		Run cuffdiff to identify differentially abundant transcripts.
			<pre>
	$ cuffdiff [-p #processors -o ./output_directory –L label1,label2,etc. –T (for time series data) –N (use upper quantile normalization –compatible_hits_norm (use reference hits in normalization) –b (use reference transcripts to reduce bias, include path to file e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa) –u (improve multi-read weighting) ] <transcripts.gtf (produced by cuffcompare) sample_A_accepted_hits1.bam, sample_A_accepted_hits2.bam,etc (all produced by tophat) sample_B_accepted_hits1.bam,sample_B_accepted_hits2.bam, etc>		$ cuffdiff [-p #processors -o ./output_directory –L label1,label2,etc. –T (for time series data) –N (use upper quantile normalization –compatible_hits_norm (use reference hits in normalization) –b (use reference transcripts to reduce bias, include path to file e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa) –u (improve multi-read weighting) ] <transcripts.gtf (produced by cuffcompare) sample_A_accepted_hits1.bam, sample_A_accepted_hits2.bam,etc (all produced by tophat) sample_B_accepted_hits1.bam,sample_B_accepted_hits2.bam, etc>

	$ cuffdiff -o ./HG19/Cuffdiff_out_options_b_u_N_compatible/ -p 14 -L Control,PUF_kd --no-update-check -b /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa -u -N --compatible-hits-norm /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/cuffcompare_out.combined.gtf /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_001_trimmed/accepted_hits.bam /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_002_trimmed/accepted_hits.bam		$ cuffdiff -o ./HG19/Cuffdiff_out_options_b_u_N_compatible/ -p 14 -L Control,PUF_kd --no-update-check -b /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa -u -N --compatible-hits-norm /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/cuffcompare_out.combined.gtf /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_001_trimmed/accepted_hits.bam /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_002_trimmed/accepted_hits.bam
			</pre>

Davebrid: Pasted in Instructions from Richard McEachin

2011-09-14T13:15:57Z

Pasted in Instructions from Richard McEachin

New page

RNA-Seq pipeline – known transcripts
1. Run FASTQ to assess quality of reads from sequencer and:
a. Filter for quality, if applicable
b. Trim, if applicable
c. http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/

2. Run bowtie-build to generate Burroughs Wheeler transformed reference genome (.ebwt format).
a. http://bowtie-bio.sourceforge.net/index.shtml (bowtie, tophat, and cufflinks are here).
b. [Optional input and parameter settings are in square brackets.]
c. <Required parameters are in greater than/less than brackets.>
d. This BW transformed reference genome can be created once then used repeatedly in the future.
e. $ is the command prompt.

$ bowtie-build [-f specifies reference genome is in fasta format] <path to input reference genome (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa)> <base name for reference genome output .ebwt files (e.g hg19)>

3. Run tophat to align reads to the reference genome. I’ve included a pseudo command line as well as a “real” command line.
$ tophat [-p #processors -o ./output_directory] <./reference genome in both .ebwt and fasta formats (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19)> <reads file to be aligned (e.g. s_1_1_sequence.fastq)>
$ tophat -p 5 -o ./HG19/tophat_out_hg19_001_trimmed /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19 ./HG19/Rich_trim/A_1_16_85.fastq

4. Run cuffcompare to create .gtf format reference genome from a generic reference genome. Note that cuffcompare adds the tss_id and p_id columns that you will need in cuffdiff. This .gtf reference can be created once then used repeatedly in the future.

$ cuffcompare [-o ./output_directory] < input file twice (e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19.gtf )>

$ cuffcompare -o ./cuffcompare_out /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf /ccmb/CoreBA/BioinfCore/Common/DATA/CufflinksData_hg19/hg19_genes.gtf

5. Run cuffdiff to identify differentially abundant transcripts.

$ cuffdiff [-p #processors -o ./output_directory –L label1,label2,etc. –T (for time series data) –N (use upper quantile normalization –compatible_hits_norm (use reference hits in normalization) –b (use reference transcripts to reduce bias, include path to file e.g. /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa) –u (improve multi-read weighting) ] <transcripts.gtf (produced by cuffcompare) sample_A_accepted_hits1.bam, sample_A_accepted_hits2.bam,etc (all produced by tophat) sample_B_accepted_hits1.bam,sample_B_accepted_hits2.bam, etc>

$ cuffdiff -o ./HG19/Cuffdiff_out_options_b_u_N_compatible/ -p 14 -L Control,PUF_kd --no-update-check -b /ccmb/CoreBA/BioinfCore/Common/DATA/BowtieData/H_Sapiens/hg19.fa -u -N --compatible-hits-norm /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/cuffcompare_out.combined.gtf /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_001_trimmed/accepted_hits.bam /ccmb/CoreBA/BioinfCore/Projects/Goldstrohm_McEachin/HG19/tophat_out_hg19_002_trimmed/accepted_hits.bam