Searching for adapter sequences in FASTQ files - metgenomics

Question

I have recently received some metagenomic data from 16S rrna sequencing. The sequencing company claim to have removed primers, however not adapter sequences. Please note that the files have been demultiplexed.

In the platform, we have forward and reverse strand fastq files. For each fastq file, there is information regarding the adapter used, for each strand.

However, when I run the following command: cat myFastQ_forward.fastq | grep 'adapter_forward', it returns nothing. My question is, shouldn't the adapters be identified successfully simply by looking for an exact match in the fastq files? If not, why?

Thanks,

Answer 1

It depends on the read layout they used, but typically when I hear anyone talk about removing adapters in paired-end sequencing, it's the occurrences of adapter sequences at the ends of each read from the complementary strand's adapter. For example see the figures on this IDT page about 16S sequencing. If a given sequence is short enough/number of sequencing cycles high enough, you'll end up seeing "past the ends" on each read.

For example, with a reverse read reverse-complemented and aligned with a forward read, you could maybe see something like this off the sequencer:

                       |-->
Forward:               BarcodefwdAmpliconBarcoderevAdapterrev...
Reverse:  ...AdapterfwdBarcodefwdAmpliconBarcoderev
                                               <--|

And then if the company already removed barcodes and primers at the start of the reads, maybe they gave you:

                                 |-->
Forward:                         AmpliconBarcoderevAdapterrev...
Reverse:  ...AdapterfwdBarcodefwdAmplicon
                                     <--|

So the material you'd want to trim is on the ends, and reverse-complemented. How much you see depends on the lengths involved so maybe you'll see partial adapters or none at all, and there may be sequencing error, too. See cutadapt's documentation about trimming 3' adapters and paired-end reads for more on that.