Question: RNA-seq read alignment
0
gravatar for amy16
3.0 years ago by
amy1640
amy1640 wrote:

I tried mapping PE RNA-seq reads to the reference genome (chickpea) using hisat2 and tophat tools with default settings. I got 0.00% alignment rate. what are the main parameters that can be tweaked to get a good alignment rate.

PS: I am still a novice in this area.

hisat2 tophat • 1.6k views
ADD COMMENTlink modified 3.0 years ago • written 3.0 years ago by amy1640
1

If the alignment was truly 0% then take a sample of reads (convert them to fasta) and blast at NCBI to verify that the data is really from chickpea and not something else.

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by genomax91k
1
gravatar for amy16
3.0 years ago by
amy1640
amy1640 wrote:

What is the difference between using STAR and HISAT2 for aligning reads to reference genome after trimming adapters? Can the output files generated from HISAT2 (.sam) be used for differential gene expression analysis?

ADD COMMENTlink written 3.0 years ago by amy1640
1

This should have been posted as a separate question. Yes you can use the alignment files generated by either for DE. You will need to use featureCounts to get counts. STAR is able to generate gene counts with an option provided at run time, if you want to avoid having to run featureCounts.

ADD REPLYlink written 3.0 years ago by genomax91k
2
gravatar for Sparrow_kop
3.0 years ago by
Sparrow_kop230
China
Sparrow_kop230 wrote:

Could you post the commend line? Maybe some parameter is wrong.

ADD COMMENTlink written 3.0 years ago by Sparrow_kop230
hisat2 -p 2 -q -c -5 15 -3 5 --phred33 -t -x ht2_Ca_v.1_index -1 P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L003_R1_paired_1.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R1_paired_1.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R1_paired_1.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R1_paired_1.fastq -2 P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L003_R2_paired_2.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R2_paired_2.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R2_paired_2.fastq,P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R2_paired_2.fastq -S P1_C1_CBBYRANXX_CGGCTATG-TATAGCCT.sam --new-summary
ADD REPLYlink modified 3.0 years ago by genomax91k • written 3.0 years ago by amy1640
1

You can remove "- c" option, and try again. From hisat2 help: "-c <m1>, <m2>, <r> are sequences themselves, not files", but in your command , -1 and -2 refer to sequence file, not the sequence themselves....

"- c" can used in the below demo:

hisat2 -c -x index -1 ATCTCGTCGTCGCT -2 ATGATGATCGTGTC -S out.sam
ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by Sparrow_kop230

Yeah... -c is for the sequence themselves and not for files...

ADD REPLYlink written 3.0 years ago by amy1640
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