Dear All,
I used STAR to map paired-end reads onto the reference genome:
The mapping looks good:
![](https://imgur.com/download/OV7Ssne)
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:
First, I got a weird warning:
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! ![Confused](https://www.seqanswers.com/core/images/smilies/confused.png)
I would appreciate some hints on that.
Best wishes,
Staszek
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 \
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
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.
![Confused](https://www.seqanswers.com/core/images/smilies/confused.png)
I would appreciate some hints on that.
Best wishes,
Staszek
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