Mapping paired-end reads to a draft genome using HISAT2
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2.8 years ago
Farbod ★ 3.3k

Dear Biostars, Hi

I have the RNA-seq data of a salmon fish (3 cond1 and 3 cond2 as biological replicate and both are paired-ends -> 12 fastq files ) and I have done Trinity de novo assembly and DEG analysis on them.

Recently the draft genome of that salmon species have been released.

I want to run a genome-guided and then DEG analysis on the same data to compare the results.

Using many helps from Biostars and @Kevin Blighe, I have selected HISAT2, StringTie approach for this purpose.

First, I created an indexed genome using: ./hisat2-build -p 6 '/home/Salmon-genome/GCF_salmon_genome.fna' ht2_base_salmon_genome

and now I have 8 *.ht2 files and want to map my reads to that reference.

Q1: Please check below mapping script I have found from here to see if it is correct for my case or not. specially I need help about paired-end data mapping. what should I write instead of sample_1.fastq in this case that I have paired-end reads?

./hisat2 -p 6  -x path/to/reference/fileName -1 path/to/fastqFile/sample_1.fastq -2 path/to/fastqFile/sample_2.fastq -S /path/to/outDir/fileName.sam &> /path/to/outDir/fileName.sam.info

Q2: can I map all my 12 files to the reference genome in one script? how? (is it by using && and repeating the script, changing fastq file names?).

Thanks

RNA-Seq alignment HISAT2 genome guided • 2.6k views
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2.8 years ago
GenoMax 100k

Q1: Sample_1.fastq will be replaced by R1 data file. Sample_2.fastq will be replaced by R2 data file.

Q2: You could follow parallel approach (Script to run blast locally with multiple files in a directory as queries ) or use this method Hisat2 multiple paired end reads

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Dear @genomax, Hi and thank you. I guess all other parameters in the script were fine.

would you please explain more what do you mean by "R1 data file" ?

My fastq files names are as :

J1-left.fq, J1-right.fq, J2-left.fq, J2-right.fq, J3-left.fq, J3-right.fq, H1-left.fq, H1-right.fq, H2-left.fq, H2-right.fq, H3-left.fq and H3-right.fq.

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left is likely equivalent to R1 and right should be R2. You can look in the read headers of left and right files. You may see something like

R1 - @EAS139:136:FC706VJ:2:2104:15343:197393 1:Y:18:ATCACG

R2 - @EAS139:136:FC706VJ:2:2104:15343:197393 2:Y:18:ATCACG
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Hi there, I have used "./hisat2 -p 6 -x ht2_base_salmon_genome -1 '/media/Seagate Backup Plus Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_left.fq' -2 '/media/Seagate Backup Plus Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_right.fq' -S H1.sam &> H1.sam.info" and then I received this warning in the H1.sam.info file:

Warning: Same mate file "/media/Seagate" appears as argument to both -1 and -2

Extra parameter(s) specified: "Backup", "Plus", "Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_left.fq",

"Backup", "Plus", "Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_right.fq" 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) Command:

/home/Desktop/salmon-genome-2018/hisat2-2.1.0/hisat2-align-s --wrapper basic-0 -p 6 -x ht2_base_salmon_genome -S H1.sam -1 /media/Seagate -2 /media/Seagate Backup Plus Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_left.fq Backup Plus Drive/Bioinformatics/RNA-Seq/RNA_Seq_Data/clean_reads/H1/H1_clean_right.fq (ERR): hisat2-align exited with value 1

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You would be wise to remove spaces in file/directory paths. I suggest you rename Seagate Backup Plus Drive as Seagate_Backup_Plus_Drive

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I know that this is not a bioinformatic question but do you have any idea how I can change the name of my external hard drive in Ubuntu 14.04?

I tried and get this error :

Cannot change label on mounted device of type filesystem:ntfs. (udisks-error-quark, 11)

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See if this helps: https://askubuntu.com/questions/731579/problem-in-renaming-the-partition-and-opening-a-file-through-terminal

If your drive is formatted using ntfs (windows file system) then are you able to write to it under ubuntu? As I recall that requires installation of some additional software (which you may have done).

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