Hello everyone,
I have pair-end reads (2x300) from V4 16S region (515F 5′-GTGCCAGCMGCCGCGGTAA and 806R- 5′-GGACTACVSGGGTATCTAAT). I have 5 samples and 2 reads in fastq format (R1 and R2) for each sample. I noticed that the reads have primers and barcodes inside them, as shown in the attached figure. My question is if It is mandatory to remove only the barcodes or both primers and barcodes? How does it affect that I do not remove primers or barcodes sequences from the reads before starting the analyzes in qiime 2? I would really appreciate any help.
Yes! Any sequence which is not the natural sequence of the amplicon must be removed, as it will cause problem in similarly matrix of the sequences. I dont see much of a problem if you keep the primer sequence, since it is a part of amplicon. Barcodes are normally removed by the sequencing facility when they perform the demultiplexing, but might differ among different companies. The main purpose of cutadapt or similar program is basically the quality filtering which also remove the adapters/primer regions.
Yes, the primers and barcodes should be removed. If you don't remove them, the alignment to the reference genome will be messy.
Cristian Zimbru
Hi, primer is a part of a biological sequence. Why did you suggest it to remove?
Mehrdad Rabiei
For example, primers can affect the frequency of genetic variants. Especially in low coverage. For a clean data analysis, I recommend using only the trimmed sequences.
Cristian Zimbru
Do not you think the divergent and variants are the basis of 16S gene analysis?
In my idea, the presence of primer in the PCR product has a couple of advantages.
-Increase the coverage by making the sequences slightly longer.
-Increase the divergent
-annotation accuracy is improved
I would be happy to hear from you.
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