hisat2 multiple thread usage

[ronaldrcutler]

Hello all, I am trying to switch over to using hisat2 instead of tophat. I have a couple questions:

1. It seems that we are able to input a list of mates for -1 & -2. Would this be referring to inputing multiple paired reads of the same sample in to be aligned at once? For example I have 8 .fastq files (4 paired reads) - how would the input look like?

2. How do I create a specific output file and directory? Would this be using the -S flag and inputing a file name after that? I see that the default output is .sam, is there a way to output .bam files?

3. I tried to use the multithread option, -p/--threads 4, (as I have 4 cores) but got this error: -p/--threads arg must be at least 1. Any help with this?

[dpryan]

1. hisat2 -x something -1 sample_1_1.fq,sample_1_2.fq -2 sample_2_1.fq,sample_2_2.fq
2. Redirect output to a file in a directory that's already created. Hisat2 won't create directories for you.
3. Use "-p 4" or "--nthreads 4". The "/" in the documentation indicates that things on either side are the same.

[ronaldrcutler]

Thank you very much. Just to be clear:
1. This means that multiple lanes in each sample can all be run at once?
2. To redirect output to a file in a directory I would use: -S Results.sam? Any way to output a .bam, or would I just have to use samtools to do this?
3.This will make things go much faster, thanks. What is your opinion at running multiple instances of hisat2 (on separate command line windows) at once to run multiple samples simultaneously?

[dpryan]

1. Yup
2. hisat ...options... | samtools -Sbo output.bam -
3. We commonly run a few instances at once with 20-30 threads each (on cluster nodes with 64 cores each). At some point you'll saturate disk I/O, but it'll probably take a while.

[ronaldrcutler]

So I have been trying to run multiple lanes within a sample on hisat2 using this command:

hisat2 -q  -p 4 -x Xenopus_Laevis -1 /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L005_R1_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L006_R1_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L007_R1_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L008_R1_001.fastq -2 /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L005_R2_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L006_R2_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L007_R2_001.fastq, /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L008_R2_001.fastq -S Sample_1_hisat2_results.sam

These are paired reads as R1 for each read indicates mate 1 and R2 indicates mate 2. When I try and run this, I am getting this error:

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.
Error: Encountered internal HISAT2 exception (#1)

This doesn't make sense as these are all mates and not single reads. Any help with this?

[GenoMax]

Pretty sure you can't have spaces between the R1/R2 read file names.

Try

hisat2 -q  -p 4 -x Xenopus_Laevis -1 /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L005_R1_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L006_R1_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L007_R1_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L008_R1_001.fastq -2 /Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L005_R2_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L006_R2_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L007_R2_001.fastq,/Volumes/cachannel/RNA_SEQ/Notch_RNASeq/in_silico_test/Sample_1/Results_1_ATCACG_L008_R2_001.fastq -S Sample_1_hisat2_results.sam

[ronaldrcutler]

Works perfectly, thanks a lot.