Choosing sequence length values from the quality score plots

Hi everybody! I'm new to Qiime, and I have a problem that has been bothering me for a long time.
When I deblur, I don't know how to choose the amount of trim length. Can anyone enlighten me on the steps to choose the trim length? Here I attach my result of demux.qzv


Hi @nur.azizah93,
These quality scores look funky! It looks like you have paired end sequences have been merged together, but were merged on the wrong sides of the sequences.

Can you tell me a bit more about any data preparation that was preformed before your data was imported into qiime2?

1 Like

Hi Chloe, thanks for your response. I already found my mistakes. So, the sequence is my practice file downloaded from GenBank Sequence Read Archive, and I forgot to split the file when downloading. Here is my new result of demux.qzv.

After that, I trimmed the sequence using cutadapt trim. Next, I will perform deblur denoise-16s. Can you help me find the right command and trim length?

I got this command from my lecturer, but He didn't explain how to find the right values of trim length and job to start. So, I am expecting your help. Thank you.

qiime deblur denoise-16S
--i-demultiplexed-seqs trimmed.qza
--p-trim-length 455
--o-representative-sequences rep-seqs.qza
--o-table table.qza
--p-jobs-to-start 5
--o-stats deblur-stats.qza

Hi @nur.azizah93,

I suggest arranging a meeting with your lecturer to learn more about the data and the decision process that they would take to select trimming settings. Discussion in-person with your lecturer is the best way to learn from their decision process, which after all might differ from the answers that we can provide.

I also suggest reading our online documentation in more detail, as the commands and parameters are described in quite some detail there. Additionally, I would suggest looking at our youtube videos:

Selecting trimming settings is a highly selective process. It depends in part on your own experimental goals! So I suggest reading more about this in the documentation and see also our code of conduct related to this point: Code of Conduct - QIIME 2 Forum

I am moving this to a "general discussion" topic, in case others on the forum wish to provide input. But consulting on exact trimming parameters is not a "user support" topic.


Hai Everyone. I obtained the raw file (paired-end) from Gen Bank SRA. I am still a newbie in using QIIME2.

I already demultiplexed the data, trimmed the primers and adapters, and joined paired ends using vsearch. After that, I tried to denoise using Deblur. However, I need clarification about the correct trim length that I should use. I have tried different values, resulting in different taxa counts. For example, when I used trim length 150, the result was 55 taxa. Another time, I used 279, resulted in 65 taxa. I hope anyone can give me some suggestions to solve this.

Here is the result of demux joined.

Thank you for helping.

Hi @nur.azizah93,

I merged this post back into our original discussion because we try to keep related post on the forum in the same thread.

As I said earlier, deciding trimming settings is really variable for each analysis and it is up to you to decide what is reasonable for your data. Again, I would really recommend the qiime2 youtube channel for video content that goes into detail about how to decided trim length.

So I am going to ask some questions and hopefully that will help illuminate what needs to be considered when deciding trim lengths.

  1. Why is trimming your sequences necessary?
  2. At your chosen trimming length, are you getting good results? (Check out your deblur-stats)
  3. When you trim at 150, what length of sequence are you getting?
  4. What benefits might you get if you have a longer sequence?

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.