Please help me！I use DADA2 to generate ASV table error, request your help!

lizgehret · June 8, 2022, 5:44pm

Welcome to the forum!

This is the the issue - which could be caused by a few different things:

Having a differing number sequence counts between your forward and reverse reads.
The sequences in one of the two paired files could be out of order with respect to the other.
There could be non standard headers in one (or both) of the files that makes the parser "think" the files are mismatched.

This is actually not an uncommon issue - I've linked a few forum posts below, as well as a Github issue that will provide you with a handful of troubleshooting steps you can take to determine the source of this mismatch and how to resolve it.

github.com/benjjneb/dada2

Mismatched forward and reverse reads

opened 05:00PM - 22 Apr 19 UTC

closed 12:27AM - 23 Apr 19 UTC

emilyjunkins

Hello, I am getting the following error: `Error in fastqPairedFilter(c(fnFs[…i], fnRs[i]), c(filtFs[i], filtRs[i]), : Mismatched forward and reverse sequence files: 7483, 3490.` I used q1 to demultiplex my reads using the suggested parameters. I added the `_R1` or `_R2` to each file. The following is my dada2 script: `path <- "/home/ocho/RC2PWPCR_ASVs/qiimedemux" fns <- list.files(path)` `fastqs <- fns[grepl(".fastq$", fns)] fastqs <- sort(fastqs)` `fnFs <- fastqs[grepl("_R1", fastqs)] fnRs <- fastqs[grepl("_R2", fastqs)]` `sample.names <- sapply(strsplit(fnFs, "_"), `[`, 1)` `fnFs <- file.path(path, fnFs) fnRs <- file.path(path, fnRs)` `filt_path <- file.path(path,"filtered") if(!file_test("-d", filt_path)) dir.create(filt_path)` `filtFs <- file.path(filt_path, paste0(sample.names, "_F_filt.fastq")) filtRs <- file.path(filt_path, paste0(sample.names, "_R_filt.fastq"))` #i need to remove the barcode (12) + the linkerprimer (37) AND the R primer (20) #because there is only 9bp overlap here, I cannot truncate `for(i in seq_along(fnFs)) { fastqPairedFilter(c(fnFs[i], fnRs[i]), c(filtFs[i], filtRs[i]), trimLeft = c(49, 20), truncLen = c(239, 251), maxN = 0, maxEE = 2, truncQ = 2, verbose = TRUE)` And I received the above error message. I am not quite sure the issue as I have prepared sequences this way before (using q1 demultiplexing algorithm). To verify, I also used adapter removal's demultiplex algorithm as well and it was the same error. The files that it gets hung on are are also the first in the lists (samples `10A_R1.fastq` and `10A_R2.fastq`). I have seen this problem mentioned [here](https://github.com/benjjneb/dada2/issues/261) but couldn't fit the solutions to my issue and also on q2 where it seemed to be a file naming [issue](https://forum.qiime2.org/t/dada2-mismatched-fwd-and-rev-reads/2133). Thanks!

Hope this helps!

Cheers