Go Back   SEQanswers > Bioinformatics > Bioinformatics

Similar Threads
Thread Thread Starter Forum Replies Last Post
Strands after fragmentation. Step 1. Gaurav_genomics Illumina/Solexa 0 05-15-2016 06:42 PM
Question about genotype and +/- strands leontp587 Bioinformatics 3 07-02-2014 06:02 AM
mate strands in IGV tahamasoodi Bioinformatics 2 03-11-2013 09:22 PM
tophat junctions strands doublehelix82 Bioinformatics 0 08-11-2012 01:03 PM
Why Do a Gene's Reads Appear on Both Strands? CompBio General 6 10-05-2009 10:01 PM

Thread Tools
Old 12-12-2018, 12:29 PM   #1
Junior Member
Location: Poland

Join Date: Dec 2018
Posts: 2
Default cufflinks does not understand strands?

Dear All,

I used STAR to map paired-end reads onto the reference genome:

~/apps/STAR/bin/Linux_x86_64/STAR --runMode alignReads \
				  --runThreadN 10 \
				  --genomeDir ../../../genome/GCA_..../ \
				  --readFilesIn R1.fastq R2.fastq \
				  --outFilterIntronMotifs RemoveNoncanonical \
				  --outSAMattrIHstart 0 \
				  --alignIntronMax 1 \
				  --alignMatesGapMax 200 \
				  --outFileNamePrefix ./out/ \
				  --limitBAMsortRAM 1048477838 \
				  --peOverlapNbasesMin 0 \
				  --quantMode GeneCounts \
The mapping looks good:

Note that reads are colored according to the "first-of-pair strand" rule.

The next step is reference-guided transcripts reconstruction. To this end, I tried to use cufflinks, but without success:

~/apps/cufflinks-2.2.1.Linux_x86_64/cufflinks -o ./out/ \
					      -p 10 \
					      --library-type fr-firststrand \
					      -g ../../../genome/GCA_XXX/GCA_XXX.gff \
First, I got a weird warning:

Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges.  It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
Second, the predicted transcripts look odd. For example, the two genes (gene0 and gene1 - see image above) were merged into a single transcript, despite they have the opposite orientation!

I would appreciate some hints on that.

Best wishes,
staszekdh is offline   Reply With Quote
Old 12-18-2018, 05:49 AM   #2
Junior Member
Location: Poland

Join Date: Dec 2018
Posts: 2

Problem solved. I used HISAT2 with "--rna-strandness RF" + stringtie and now I get reasonable results.


$HISAT2_HOME/hisat2 -q \
                -p 10 \
                -x ../../../genome/genome/XX \
                -1 ../B1R_S1_R1_001.clean.fastq \
                -2 ../B1R_S1_R2_001.clean.fastq \
                -S out_mock_rep1_clean/alns.sam \
                --fr \
                --rna-strandness RF \

~/apps/stringtie-1.3.5.Linux_x86_64/stringtie \
	../hisat/out_mock_rep1_clean/alns.sorted.bam \
	-o ./out_mock_rep1_clean/out.gtf \
	-p 10 \
	--fr \
        -G ../../../genome/xxx.gff
BTW: I found this post useful
staszekdh is offline   Reply With Quote

cufflinks, paired-end sequencing, rna-seq, star

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 11:35 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2019, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO