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SDPA_Pet 08-16-2017 11:39 AM

Should I worry about the direction of my reads
 
Hello, I recently got my metagenomic illumina sequencing back (hiseq). There is one sample that I would like to do some blast. Since it was sequenced using paired-end sequencing (I have two fastq files forward and reverse sequencing) for this sample, here is what I am going to do.

First, I interleaved the two fastq files using bbmap. So, I will get one fastq file and easy for me. Next, I plan to use the file to do blast.

Here is my concerned. When I run BBmap, I didn't notice if there is no option to sort the direction of the two fastq files. For example, I want to make all reads in the interleaved fastq file have the same direction (e.g., 5'-3' or 3'-5'). I am concerned if this interleaved fastq file mixed with reads having different directions could make the NCBI blast program failed to find the best match in the database.

Here are my questions:

1>The reads in the two fastq files (forward and reverse) that I got from the sequencing center are same directions or opposite directions? I know the machine sequenced from opposite direction. How about the reads directions in the fastq files? Already sorted? How can you tell? Are they from 3' to 5' or 5' to 3'?

2>Are the common software (e.g., BLAST+, RApsearch, DIANMOND) consider the direction problem, when they do search?

Thanks

kmcarr 08-17-2017 06:35 AM

Unless it is explicitly stated that a reported DNA sequence is presented in a 3' to 5' orientation it is accepted convention that all DNA sequences are written 5' to 3'. All sequence data reported by an Illumina (or other) sequencer is in 5'->3' orientation. BLAST or any other sequence alignment program expects and assumes that your input FastQ files are 5'->3'. These programs all internally manage the alignment of the query to the reference in both the its input orientation and its reverse complement.


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