SAM file generation problem

Question

[kuhelikadas1995]

my SAM files generated are of 1.1kb each.. also the alignmnt is not done.. what to do??

[finswimmer]

Please put some more effort into your questions to get proper help.

• What programs are you using?
• Show us the complete commands you were using.

[kuhelikadas1995]

nohup hisat2 --dta -cufflinks -x ../5.Reference_genome/genome -1 ../4.Trimmed_fastq/SRR1131659_Trimmed_1.fastq.gz -2 ../4.Trimmed_fastq/SRR1131659_Trimmed_2.fastq.gz -S SRR1131659.sam -p 20 &

this was the command used for aligning the adapter trimmed fastq files against human refernce genome. the SRA data here is paired end rna seq data. Command used for adapter trimming is:

cutadapt -a AGATCGGAAGA -A AGATCGGAAGA -o SRR1131659_Trimmed_1.fastq.gz -p SRR1131659_Trimmed_2.fastq.gz -m 20 SRR1131659_R1.fastq.gz SRR1131659_R2.fastq.gz -q 30

[manuel.belmadani]

In the future when you paste code examples, highlight your code and click the "101 010" button to make it a code block, makes it easier to read.

[kuhelikadas1995]

done. but it didnot work. same problem. no alignmnt done.

[GenoMax]

If it did not work then why did you accept the answer below?

[kuhelikadas1995]

I accepted the answer nd tried.. then saw that i didnt work..

[manuel.belmadani]

Can show us the file sizes for your original and trimmed data, as well as your genome? One thing I notice is that in your cutadapt command, the quality filter (-q 30) is after the input files whereas in the documentation it's typically before.

If your trimmed files are suspiciously small I would guess that's the error.

[kuhelikadas1995]

raw data for r1 read is of size 502.3MB and that of r2 is of 498.8MB.. trimmed data for r1 is of 489.6MB and that of r2 is 486.6MB.. moreover, applying

-q 30before or after made no difference. Size of reference genome is 1.1GB(zipped file) and 4.5GB(when unzipped)

[GenoMax]

As I recall HISAT2 is one of those programs that is sensitive to order of program options and input files. So you may want to pay some attention to that order. Did you generate the indexes yourself and are they known to work?

[kuhelikadas1995]

yeah.. and it did generate the indexes for genome by myself.. nothing was mentioned about the adapteer sequence.. it used AGATCGGAAGA.. for both forward and reverse end reads. should i use AGATCGGAAGAGC as the adapter sequence fr both the end reads??

[GenoMax]

Specifying an incorrect adapter sequence should have no impact on being able to produce a good SAM file from HISAT2. It sounds like that is where your problem lies. Have you looked at the log file for HISAT2 to see if there are any errors that can be addressed?

[kuhelikadas1995]

initially i had used the command

cutadapt -a AGATCGGAAGA....and so on I mean i skipped the "-A adapter2" part keeping rest of the command same , initially for trimming.. but that command is for single end data..whereas my data is of paired end..That time SAM files generated were of 4.4GB each or more ..and BAM files were of around 1.2GB each. but problems started showing up (SAM files are being generated of 1.1kb each..and BAM files stop being executed after 800 bytes or so) when i used the cutadapt command mentioning both adapters 1 and 2;(the cutadapt command shown in the very 1st comment of mine)as shown below:

> cutadapt -a AGATCGGAAGA -A AGATCGGAAGA -o SRR1131659_Trimmed_1.fastq.gz -p SRR1131659_Trimmed_2.fastq.gz -m 20 SRR1131659_R1.fastq.gz SRR1131659_R2.fastq.gz -q 30

Answer 1

[manuel.belmadani]

Change --dta -cufflinks to --dta-cufflinks.