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Using dada2 for single end reads #795
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Yes. Everything will run the same as the tutorial workflow except skip the reverse read parts and read merging, and form the sequence table out of the |
Thanks a lot!
…On Thu, Jun 20, 2019 at 12:36 PM Benjamin Callahan ***@***.***> wrote:
Yes. Everything will run the same as the tutorial workflow except skip the
reverse read parts and read merging, and form the sequence table out of the
dadaFs object, i.e. makeSequenceTable(dadaFs).
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Dear Ben,
Thank you very much, |
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Dear Ben, |
Yep, no different really. |
Great! Thank you so much for this swift response. Works perfectly. |
Dear Ben, error_plots_raw.pdf |
In general, subsetting data or choosing samples of better quality or lower complexity is a valid approach to 454 data often had a pretty flat error-profile because of the error modes of that technology which were dominated by indel errors. |
Thank you so much Ben for helping me to understand. |
That was typical for 454 data in my experience.
Yes, having a good model fit is the first thing to look for. The second is that the overall model looks like what is expected from that sequencing technology. |
Great! Thank you so much! |
Hi there, out <- filterAndTrim(fnFs, filtFs, I have tried increasing the maxEE value, but this didn’t make a difference. I am unsure which other parameters could be changed, or what to change them to? Thank you, |
That flag is requiring each read to be at least Infinity bases long to be kept. AKA no reads will pass the filter. |
Thank you so much! |
Dear Ben, Also, I would like to apply the collapseNoMismatch function to my data. I got ITS sequences of variable length (ca. 600-300) with a minLength set to 300bps. Would you recommend to apply this function? Thank you! |
It exists in a special class of algorithmic arguments that you can inspect using the
Was the data trimmed to span from the forward to reverse primers? If so, then the length variation is biological, and doesn't require |
Great! thank you so much!. The lenght variation is technical! |
Dear Ben, I noticed that the sum of the number of total final reads obtained in each run (run 1+run2+run3= 231499 reads) was lower that the total reads obtained in the merged table (= 198723 reads), so the merged table had 15% less reads. The merged table contained all the samples included in the 3 runs, so no problems with samples names…is there any explanation for this? |
I think the reason is that you removed chimeras on the merged table? Prior to chimera removal and immediately after |
That's the problem. The numbers are not equal immediately after "mergeSequenceTables" (prior to chimera removal). |
Can you isolate the code that leads to this issue, including the output at each step? I.e. post the code that counts the reads in each table, then the code to merge them, then the code that counts the reads in that merged table. Include the outputs, and any other messages that arise. |
Dear Ben, Sorry for any inconvenience and thanks for your time. |
The sum of the reads should be the same.
Correct. My concern now is actually that so few (1206-1185=21) common ASVs are being found between your 3 runs. Is that expected? Or am I misinterpreting the numbers you posted? |
Hi Ben, I am new in bioinformatics and I am running my first samples. I have been having a problem with the quality of my data where the reverse reads are very bad. I tried to find some explanation on the internet that would explain to me why only the reverse reads are bad. Would you mind briefly explaining to me that, if you know the answer? Or perhaps send me some links of articles where I could find this info? Thank you very much for your help. |
@aff30 I don't have any documentation to send you, but it is broadly understood that the reverse reads on Illumina are typically of worse quality, and that depending on how the library is prepared and the instrument is performing, they can be much worse. So what you are seeing isn't that unusual, although it would make me reach out to the sequencing provider and start to consider alternatives. |
Yeah, I also did not find anything, but thank you very much for your answer! |
Can I use dada2 for analysis if I have single end reads?
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