Question: Running STAR aligner on paired-end reads as single-end read
1
gravatar for Glory Basumata
15 months ago by
India/Guwahati/Indian Institute of Technology Guwahati
Glory Basumata140 wrote:

Hi, I am just curious. Can we use STAR aligner to align the paired-end reads as a single-end? What are the consequences of the output of such alignment?

ADD COMMENTlink modified 15 months ago by h.mon28k • written 15 months ago by Glory Basumata140
5
gravatar for h.mon
15 months ago by
h.mon28k
Brazil
h.mon28k wrote:

Yes, STAR can align paired-end reads as single-end, just run STAR two times, one with --readFilesIn R1.fastq, another with --readFilesIn R2.fastq.

What are the consequences of the output of such alignment?

First and foremost, the mapped reads will be considered as single-end. In addition, the proportion of multi-mappers may increase.

ADD COMMENTlink written 15 months ago by h.mon28k

Hi, h.mon, Thank you for your answer. When you say, run STAR two times, do you mean that I take one Fastq file and run it two times? First with Forward option followed by the Reverse alignment option? Thank you.

ADD REPLYlink written 15 months ago by Glory Basumata140
1

First with Forward option followed by the Reverse alignment option?

STAR doens't have Forward or Reverse options.

Assuming you do have paired-end sequencing, your fastq files may be organized in two ways:

  • separated in two files, with forward and reverse reads in each file, commonly name_R1.fastq and name_R2.fastq, or some variation of this. This is the more common scenario.

  • forward and reverse reads in one file, usually referred to as a interleaved fastq file. This is not so common.

If you have your paired-end reads separated in two files, you have to run STAR twice, once for each file. If you have your paired-end reads in a interleaved fastq file, you can just supply the name of the file and STAR will map it as single-end, as STAR doesn't recognize interleaved files (nor have a parameter to toggle interleaved ON), so the reads will be mapped as single-end.

ADD REPLYlink modified 15 months ago • written 15 months ago by h.mon28k

Hi h.mon, Thank you for the detailed explanation. I appreciate your help. Sorry for the late response, as I was doubtful when you mentioned that STAR does not have the option to align forward or reverse reads. So I have to take some time and do my research on it. What raised my doubt, initially, was the galaxy project's option for RNA STAR which asks for reads - forward and reverse

https://ibb.co/f18YRU

STAR align option

ADD REPLYlink modified 15 months ago • written 15 months ago by Glory Basumata140
3

Sorry, my explanation was a bit shabby. STAR can accept single-end reads, or paired-end reads in two files. But unlike some programs, where one has parameters for each file separately (e.g. HISAT2, with -1 pair_1.fastq -2 pair_2.fastq), STAR has only one parameter (--readFilesIn), which will behave differently if you pass one or two files to it.

If you pass one file, STAR will consider these as single-end reads: --readFilesIn single_reads.fastq.

If you pass two files, STAR will consider these as paired reads: --readFilesIn pair_1.fastq pair_2.fastq.

Galaxy wraps the command with a web front-end interface, hiding the underlying command-line from users. This means the Galaxy interface can ask for two files separately, but when STAR is run on the server, the argument passed will be --readsFiles pair_1.fastq pair_2.fastq.

ADD REPLYlink written 15 months ago by h.mon28k

This beautifully crafted answer now clears my doubt altogether in relation to supply of read inputs into STAR or Hisat2. Thank you for your help. Appreciate it.

ADD REPLYlink written 15 months ago by Glory Basumata140
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