Hi everyone,

I am new to the NGS. For training, I used paired-end data from public files, and usually, there were only two fastq files for the two reads.

Now, I receive the fastq files for my ChIP-seq studies. We did 50 bp paired-end. And for one PE file, there are four fastq files. For example:

R1F=IN1_S1_L001_R1_001.fastq
R1R=IN1_S1_L001_R2_001.fastq
R2F=IN1_S17_L002_R1_001.fastq
R2R=IN1_S17_L002_R2_001.fastq

I am now a bit confused about how to handle these 4 data.

I tried to do bowtie2 paired-end mapping for R1F as -1 and R1R as -2 but it seems not working.

Bowtie2 notification:
Note that if <mates> files are specified using -1/-2, a <singles> file cannot
also be specified. Please run bowtie separately for mates and singles.
Error: Encountered internal Bowtie 2 exception (#1)

I am also unsure about the use of Paired-end merge tools.

Please help. Thank you
Hi! I think what you have there is 2 fastq files for the same sample, each one coming from a different sequencing lane (note that some of the files have L001 and some others L002). What you need to do is to concatenate the sequencing lanes into one file and then align with the resulting file. 

    cat IN1_S1_L001_R1_001.fastq IN1_S1_L002_R1_001.fastq > IN1_S1_concatenated_R1_001.fastq
    cat IN1_S1_L001_R2_001.fastq IN1_S1_L002_R2_001.fastq > IN1_S1_concatenated_R2_001.fastq

The resulting files are the 2 mates (R1 and R2) that you want to feed into your favorite aligner.

Hope it helps!