How to align single end multiple lane data using Hisat2?

Question

[AANCHAL]

I have 4 lane , single end reads fastq files per sample that I need to align. How can I combine the 4 lanes to give as input?

I am trying to give the following command:

hisat2 -p 12 -x ./MM10_Index_HISAT/mm10 -U ./Rane_data/Acrolein-1_S4_L1_R1.fastq,./Rane_data/Acrolein-1_S4_L2_R1.fastq,./Rane_data/Acrolein-1_S4_L3_R1.fastq,./Rane_data/Acrolein-1_S4_L4_R1.fastq --rna-strandedness F -S ./HISAT_output

Also, how can I give all samples together in one command? I have 6 samples.

[Ram]

Please read the POSTING GUIDE before you Submit a new post. It's shown above the editor when you create a post. Also, please use the formatting bar (especially the code option) to present your post better. You can use backticks for inline code (`text` becomes text), or select a chunk of text and use the highlighted button to format it as a code block. If your code has long lines with a single command, break those lines into multiple lines with proper escape sequences so they're easier to read and still run when copy-pasted. I've done it for you this time.

[AANCHAL]

Thank you. This was my 1st post. Will take care of it from next time.

Answer 1

[ATpoint]

Yes, that is correct, see manual.

-U <r>

Comma-separated list of files containing unpaired reads to be aligned, e.g. lane1.fq,lane2.fq,lane3.fq,lane4.fq. Reads may be a mix of different lengths. If - is specified, hisat2 gets the reads from the “standard in” or “stdin” filehandle.

[AANCHAL]

I tried running my all sample files comma separated and it gave me an error. Is there a way to combine all samples?

[ATpoint]

and what is the error?

[AANCHAL]

Error: Encountered internal HISAT2 exception (#1)

Command: /usr/local/hisat2/hisat2-2.1.0/hisat2-align-s --wrapper basic-0 -p 12 -x ./MM10_Index_HISAT/mm10 --rna-strandedness F -S ./HISAT_output -U ./Rane_data/Acrolein-1_S4_L1_R1.fastq,./Rane_data/Acrolein-1_S4_L2_ R1.fastq,./Rane_data/Acrolein-1_S4_L3_R1.fastq,./Rane_data/Acrolein-1_S4_L4_R1.fastq,./Rane_data/Acrolein-2_S5_L1_R1.fastq,./Rane_data/Acrolein-2_S5_L2_R1.fastq,./Rane_data/Acrolein-2_S5_L3_R1.fastq,./Rane_data/Acro lein-2_S5_L4_R1.fastq,./Rane_data/Acrolein-3_S6_L1_R1.fastq,./Rane_data/Acrolein-3_S6_L2_R1.fastq,.//Rane_data/Acrolein-3_S6_L3_R1.fastq,./Rane_data/Acrolein-3_S6_L4_R1.fastq,./Rane_data/Control-1_S1_L1_R1.fastq,./R ane_data/Control-1_S1_L2_R1.fastq,./Rane_data/Control-1_S1_L3_R1.fastq,./Rane_data/Control-1_S1_L4_R1.fastq,./Rane_data/Control-2_S2_L1_R1.fastq,./Rane_data/Control-2_S2_L2_R1.fastq,./Rane_data/Control-2_S2_L3_R1.fa stq,./Rane_data/Control-2_S2_L4_R1.fastq,./Rane_data/Control-3_S3_L1_R1.fastq,./Rane_data/Control-3_S3_L2_R1.fastq,./Rane_data/Control-3_S3_L3_R1.fastq,./Rane_data/Control-3_S3_L4_R1.fastq (ERR): hisat2-align exited with value 1

[Ram]

Acrolein-1_S4_L2_ R1.fastq,./Rane
-----------------^

There are spaces in filenames in your command. Are those copy-paste artifacts?

[AANCHAL]

these are copy-paste artifacts

[swbarnes2]

Did you put spaces after the commas? Also, are you completely sure that you want to align Acreolein-2 and Acreolein-3 together?

[AANCHAL]

I want to do all in one go just like STAR if possible. STAR takes up all input files in the command line.

[AANCHAL]

hisat2 -p 12 -x ./MM10_Index_HISAT/mm10 -U ./Rane_data/Acrolein-1_S4_L1_R1.fastq,./Rane_data/Acrolein-1_S4_L2_R1.fastq,./Rane_data/Acrolein-1_S4_L3_R1.fastq,./Rane_data/Acrolein-1_S4_L4_R1.fastq --rna-strandedness F -S ./HISAT_output

Right now this is my command line I am giving and it is also giving me error:

Error:

Encountered internal HISAT2 exception (#1)

Command:

/usr/local/hisat2/hisat2-2.1.0/hisat2-align-s --wrapper basic-0 -p 12 -x ./MM10_Index_HISAT/mm10 --rna-strandedness F -S ./HISAT_output -U ./Rane_data/Acrolein-1_S4_L1_R1.fastq,./Rane_data/Acrolein-1_S4_L2_
    R1.fastq,./Rane_data/Acrolein-1_S4_L3_R1.fastq,./Rane_data/Acrolein-1_S4_L4_R1.fastq 

(ERR): hisat2-align exited with value 1

[AANCHAL]

I dont understand what I am doing wrong!

[AANCHAL]

Can somebody help with the error?

[Ram]

If you're working on a cluster, check with your admin if you're running out of resources or something of that sort. Also, don't do this constant follow up commenting, it's rude.

Answer 2

[swbarnes2]

With single end reads, you could also do something like

cat *.fastq.gz |  hisat2 -p 12 -x ./MM10_Index_HISAT/mm10 -U - --rna-strandedness F -S ./HISAT_output

[AANCHAL]

Also I downloaded the tar file of the mm10. The following are the files and contents in the mm10 folder.

total 3961472
-rw------- 1 a0malh01 unixuser  888467914 Mar 17  2016 genome.1.ht2
-rw------- 1 a0malh01 unixuser  663195880 Mar 17  2016 genome.2.ht2
-rw------- 1 a0malh01 unixuser       6119 Mar 17  2016 genome.3.ht2
-rw------- 1 a0malh01 unixuser  663195875 Mar 17  2016 genome.4.ht2
-rw------- 1 a0malh01 unixuser 1165549977 Mar 17  2016 genome.5.ht2
-rw------- 1 a0malh01 unixuser  675339278 Mar 17  2016 genome.6.ht2
-rw------- 1 a0malh01 unixuser          8 Mar 17  2016 genome.7.ht2
-rw------- 1 a0malh01 unixuser          8 Mar 17  2016 genome.8.ht2
-rwx------ 1 a0malh01 unixuser       1378 Mar 17  2016 make_grcm38.sh

Just wanted to make sure it is fine and not causing any error.

[AANCHAL]

Is this file ok? It is not causing the error mentioned above?