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