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Old 12-12-2018, 01:29 PM   #1
staszekdh
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:

Code:
~/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:

Code:
~/apps/cufflinks-2.2.1.Linux_x86_64/cufflinks -o ./out/ \
					      -p 10 \
					      --library-type fr-firststrand \
					      -g ../../../genome/GCA_XXX/GCA_XXX.gff \
					      ../star/out_mock_rep2_clean/Aligned.sortedByCoord.out.bam
First, I got a weird warning:

Code:
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,
Staszek
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Old 12-18-2018, 06:49 AM   #2
staszekdh
Junior Member
 
Location: Poland

Join Date: Dec 2018
Posts: 2
Default

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

HISAT2:

Code:
$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 \
                --dta
stringtie:

Code:
~/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
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cufflinks, paired-end sequencing, rna-seq, star

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