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Reference genome alignment

How to specify paired end input files from different lanes for alignment using hisat2?

I get the following error: Warning: Output file 'P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R1_paired.fastq,' was specified without -S. This will not work in future HISAT 2 versions. Please use -S instead. Extra parameter(s) specified: "P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R1_paired.fastq,", "P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R1_paired.fastq", "P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R2_paired.fastq,", "P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R2_paired.fastq,", "P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R2_paired.fastq" Note that if <mates> files are specified using -1/-2, a <singles> file cannot also be specified. Please run bowtie separately for mates and singles. Overall time: 00:00:00 Error: Encountered internal HISAT2 exception (#1)

rna-seq alignment
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written by amy16 • amy16

Is it ok to generate genome index using the default parameters available within the tool? or does it vary from case to case?

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written by amy16 • amy16

That should be fine. If you are using one of the model genomes then get the pre-made indexes from the link below.

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written by GenoMax GenoMax

The genome I need Cicer is not within that link.

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written by amy16 • amy16

Create your own in that case (instructions). 

hisat2-build ref.fa cicer_idx

use --large-index option if your genome is bigger than 4 billion bp.

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written by GenoMax GenoMax

the predicted genome size is 740 Mbp

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written by amy16 • amy16

Can u please advise me if I should go for large or small genome index for Cicer genome size of 740 Mbp?

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written by amy16 • amy16

Small genome index should be fine.

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written by GenoMax GenoMax

1 answer

Multiple files can be specified by using a comma separated list of the files for the -1 and -2 options.

hisat2 -f -x hg19_indexed -1 read1_R1.fq,read2_R1.fq,read3_R1.fq -2 read1_R2.fq,read2_R2.fq,read_R2.fq -S eg2.sam

in your case, something like this

hisat2 -f -x base_name_of_index -1 P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R1_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R1_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R1_paired.fastq -2 P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R2_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R2_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R2_paired.fastq -S P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT.sam
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written by GenoMax GenoMax

Do I just specify the file name or do I have specify the path as well?

I get this error: /tmp/slurmd/job4228023/slurm_script: line 17: P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L003_R1_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R1_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R1_paired.fastq,P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R1_paired.fastq: No such file or directory

Although it has generated the output file of 15.1GB. I would like to know if this is right.. Or something has gone wrong.

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written by amy16 • amy16

You will have to use full or relative paths for all files (if they are not in the current directory). Hard to tell if the file you generated was right. You could set a variable to point to the directory your files are in e.g. MYFLS=/full_path_to_directory_w_files and then do

hisat2 -f -x base_name_of_index -1 $MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R1_paired.fastq,$MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R1_paired.fastq,$MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R1_paired.fastq -2 $MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L004_R2_paired.fastq,$MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L005_R2_paired.fastq,$MYFLS/P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT_L006_R2_paired.fastq -S P1_C_1_CBBYRANXX_CGGCTATG-TATAGCCT.sam
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written by GenoMax GenoMax

Is it possible to use index file with .ebwt (bowtie2) extension for alignment using HISAT2 ?

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written by amy16 • amy16

Build your own HISAT2 indexes or download them from HISAT2 site (right hand column).

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written by GenoMax GenoMax

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