fastq file format

Question

[Vladimir Leshuk]

Are reads' sequences in a fastq file written in 5' to 3' direction?

Answer 1

[ATpoint]

yes

[Vladimir Leshuk]

Just to make sure... Do 5'-ends of all reads in the library correspond to the 5'-ends of the original sequence?

I have a protein that binds small DNA molecules (14-25 nt). I prepared the library of these small DNAs (which were bound by the protein) and single-end sequencing was conducted. Is 5'-end of the molecule that was associated with my protein equal to the 5'-end of the corresponding read?

[ATpoint]

So you have dsDNA as above, right? And then ligated adapters to them and amplified it with primers compatible with Illumina sequencing? In this case you will have a random mix of amplicons with the P7 and P5 ends on either the original top- or bottom strand. Hence, if you sequence this library some reads will represent the actual 5' ends of the "top" strand and others the 5' ends of the "bottom" strand. You will then need to align these reads to a reference or assemble the reads to get the consensus sequences for each of these small DNA molecules. Does that make sense?

[Vladimir Leshuk]

Yes, it makes sense. Actually, I need to estimate nucleotide bias across these small DNA molecules. If I run commands like this would I get my answer? I want to answer the question "which nucleotides does protein prefer in different positions?"

samtools fastq file.bam  >  file.fastq 
cutadapt -m 18 --length 18 --fasta file.fastq  > file.fasta
weblogo < file.fasta > logo

To make sure...:) If I retrieve sequences of aligned sequences from the bam file would they be in this form in a fastq file? And would their sequences be equal to either bottom or top strand?

Aligned to the forward strand: 5' sequence 3'

Aligned to the bottom strand: 5' sequence 3'

My protein holds 5'-end of the small DNA molecule in a fixed position and for me the 5'-side this molecule is more important. By cutadapt ---length 18 command I trim them from the 3' side to the length of 18; and by -m I discard all sequences that are less than 18 nt.

Thanks for your time and patience!!!

Answer 2

[swbarnes2]

You need to look up how your library prep works.