Number of Reads Increasing

[hamcan]

Hi there,
I trimmed my reads and then merged them using FLASh. However, I found that the number of reads before processing (trim and merge) is lower than the number of reads after processing.
Is there an explanation for this?
Thanks

[Brian Bushnell]

When you merge a read pair, you turn 2 reads into 1 read. Therefore, you should expect the number of reads to decrease.

[hamcan]

i know i should expect a decrease but i'm getting an increase. do you know why?

[Brian Bushnell]

Perhaps you can post your exact commands used at each step, as well as how you are calculating the number of reads?

[hamcan]

First step I'm doing is using Trimmomatic to trim and remove my adapters using the PE setting:

java -jar trimmomatic-0.36.jar PE File1_L001_R1_001.fastq File1_L001_R2_001.fastq /home/rachelle/File1_R1paired.fastq File1_R1unpaired.fastq File1_R2paired.fastq File1_R2unpaired.fastq ILLUMINACLIP:/usr/local/trimmomatic/adapters/NexteraPE-PE.fa:2:30:10:6 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:25 MINLEN:36

Afterwards, I take R1paired and R2 paired and merge them using FLASh with a minum of overlap of 15.
I then concatenate the out.extended frags with the two notcombined files for a final file that is then concatenated with R1UNpaired and R2UNpaired.

That final file has more reads than the beginning.
I looked into it some more and saw that my reads are increasing after FLASh. I am assuming it has to do with concatenation of the two notCombined files. Does anyone know what those two files are? And if I should be concatenating them with my out.extendedfrags file?

Thanks

[GenoMax]

Best option would be to merge first (use bbmerge.sh from BBMap) and then trim (bbduk.sh from BBMap).

The only way number of reads is going to increase is if you double dip into the read pool. You must be somehow doing that. Since your merged reads become "single-end" you should not merge them with remaining PE reads.

[HESmith]

Paired-end reads are typically counted once (unless it's the Illumina marketing team). Merged reads will also be counted once after FLASH, but unmerged reads now become two single-end reads and are counted twice.

[hamcan]

that makes perfect sense!!! Thank you so much