Mismatched R1/R2 PAIRS FOUND (these will crash filterAndTrim)

[Kumaratenna]

Hi,
When running the DADA2 pipeline for my bioinformatics analysis, I encountered an issue indicating that mismatched forward and reverse read pairs are present in my FASTQ files.

Could you please advise on the best way to resolve this issue? I would appreciate your guidance on whether this is likely due to incorrect file pairing, lane merging, or another common cause, and how I should properly fix it before proceeding with the analysis.

Thank you very much for your help.

--- Checking R1 vs R2 read counts (to detect mismatched pairs) ---

!!! MISMATCHED R1/R2 PAIRS FOUND (these will crash filterAndTrim) !!!
sample
136 LIN-25-02-136
137 LIN-25-02-069
138 LIN-25-02-070
157 LIN-25-02-089
158 LIN-25-02-090
159 LIN-25-02-091
forward
136 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-136_R1.fastq.gz
137 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-069_R1.fastq.gz
138 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-070_R1.fastq.gz
157 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-089_R1.fastq.gz
158 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-090_R1.fastq.gz
159 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-091_R1.fastq.gz
reverse
136 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-136_R2.fastq.gz
137 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-069_R2.fastq.gz
138 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-070_R2.fastq.gz
157 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-089_R2.fastq.gz
158 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-090_R2.fastq.gz
159 /home/kumaratp/Clean_files_Bacteria_2/LIN-25-02-091_R2.fastq.gz
nF nR
136 137817 186419
137 41482748 56112119
138 41482616 56112119
157 156807 93965
158 47198907 28283164
159 47198907 28283164

[benjjneb]

That you would have identical numbers of reads in samples 138/139 and 158/159 is highly suspicious if I am reading this right, and would suggest an issue with file management.

If file management is OK, then read mismatches can occur because of previous filtering/processing that treated R1 and R2 files independently. This can be fixed using the filterAndTrim(..., matchPairs=TRUE) option in the filterAndTrim function in the dada2 R package. See ?filterAndTrim for a bit more description of that argument and what it does.