Bowtie2 alignment error with different amount of reads in R1 and R2

Question

[vnewtoboiin]

Hi all, I have an issue when trying to align a ChIPseq input reads to the genome using bowtie2. The error that comes up is: Error, fewer reads in file specified with -2 Error, fewer reads in file specified with -2 than in file specified with -1than in file specified with -1

I have removed singletons using fastq_pair but it still seems as though R1 and R2 have different amount of reads... What could be the cause of this and how do I fix it?

Answer 1

[ATpoint]

How were the data processed in the first place? Something like trimming with a non paired-end aware tool?

[vnewtoboiin]

The only processing I’ve done so far is trimgalore —very sensitive and fastqc, followed by fastq_pair

[ATpoint]

TrimGalore for each file separately or in paired-end mode?

[vnewtoboiin]

For each file separately

[ATpoint]

That is the problem, this will leave singletons. TrimGalore (and cutadapt which it is a wrapper around) have paired-end support which is described in its manual, please use it to avoid asynchronous fastq files.

[vnewtoboiin]

I will give trimgalore --paired a go, thanks for your help!