Which primer should I use in fungal ITS2 region amplicon sequencing

Hi everyone !

Recently I try to characterize my soil sample with ITS2 region sequencing.
I have used primer 5.8S-Fun/ITS4-Fun from paper Accurate Estimation of Fungal Diversity and Abundance through Improved Lineage-Specific Primers Optimized for Illumina Amplicon Sequencing
which is also used in Fungal ITS analysis tutorial.
The gelelectrophoresis result is a little disappointed since the amplicon length is at least 500bp so it can be sequenced under illumina PE250 strategy.
Which primer should I choose?:expressionless:

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Goo to hear from you @sixvable

Try them both, and see how the results compare!

If you run dada2 directly in R, the mergePairs() function includes the justConcatenate=TRUE argument that will not try to merge your reads and will instead “justConcatenate” them. If you are willing to try R, this is a good option for non-overlapping reads. Unfortunately, this is not yet available in the dada2 Qiime 2 plugin. :crying_cat_face:

Colin

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Thank you @colinbrislawn

I just have another question :laughing:
If I use non-overlapping reads ,can I still assign those with same way in q2-feature-classifier or other special way.I only saw one guy using non-overlapping reads and he assign his reads1 and reads2 separately.

sixvable

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Good question.

If you have non-overlapping reads, dada2 through Qiime 2 does not allow you to process them together. So you would have to denoise them separately, then assign taxonomy to them separately. Yes, you can use q2-feature-classifier on read1 and read2 separately or paired.

You bring up a very interesting question: how do you assign taxonomy if you concatenate using mergePairs(justConcatenate=TRUE)? I’m not sure how best to classify concatenated reads, but I guess that’s not yet a problem within Qiime 2. For now, you can pair reads or process them separately.

Colin

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