Hey everyone. I have a bam file which I converted to sam (via samtools) and then used Picard's SamToFastq script and split the bam into two fastq files. I am now trying to run the de novo assembler Trinity on these files. I know they are strand specific, but I do not know their orientation. Are they forward-reverse orientation or reverse-forward? Which strand is the left and which is the right? Does anyone know how to determine this?
Sorry I meant to comment on your answer not create my own. I am not sure what you mean, unless you replace "isn't it?" with "can't you?". Here is an example, read1: @HS5_233:5:1103:12424:50065/1 GTGCAGAGGAGAACGCAGCTCCGCCCTCGCGGTGCTCTCCGGGTCTGTGCTGAGGAGAACGCAACTCCGCCGTCGCAAAGGCGCCGCGCCGGCGCAGACG + ??@DD>;BF?FHFGAFFBHED>A>@;6;1>@?9(8;/383:48>@>>>B#########################
read2: @HS5_233:5:1103:12424:50065/2 GCCGCCCCACCAACCCCCCCCCGTACGCGGGCGTCTCCGCGGCCGGGCCACCCCGCCCGCCCCCTCGACGCGCCCGCCGGAGTATCTGGTCCTGCGCCCG + =11++)0@F###########################################################################################
Are your original FASTQ files strand-specific and paired end? The FASTQ file converted back from SAM will not have any information about read orientation. Given that your reads came from SS protocol, in your sam file, the 2nd column => flag should tell you all the reads that belong in first pair of your fastq and all the reads that are in the 2nd pair of your fastq. You should split your sam files based on the flag to 2 sam files and picard samToFastq on each of them separately.
In short, post from your SAM file, a properly paired read and its pair.