Hi sir , I am a beginner so i have lots of questions in my mind for raw data analysis, i have some questions I hope you will give the answers to my queries.
i have 2 reads for each sample (R1 and R2) and one is forward and one is reverse so its paired-end sequence? am I right?
but sir i don’t have the barcode sequence and primers information of these sequences so for further analysis i choose the paired-end manifest format. but i want to try some other methods for my raw sequence. so my question is should i do denoising with separately for R1 (forward) and R2(reverse) and after that, i will merge these sequences? if your answer is yes how can we do this? i follow the FMT tutorial for my sequence but after the merging and created the final table my six samples (R1+R2) become 12. i just want to know which tutorial i follow for my R1 and R2 sequence analysis?(denoising separately first and after i can merge R1 and R2 file and make one read)
Please remember for future that there are many non-male users/moderators!
Yes, you have demultiplexed Paired-End reads.
Since your reads are already demultiplexed you don’t need barcodes. Those are simply used to map your sequences to a particular sample. You need to simply import your reads as paired-end reads and follow whichever denoising method you want, i.e ada2 or deblur. Using the manifest format import approach here is good, just make sure you use the paired-end method.
The FMT tutorial you mention deals with importing 2 separate single-end datasets and merging them. This is different than paired-end reads from 1 dataset which you have. Try the Atacama tutorial and just skip to the dada2 denoise-paired step.
Please remember for the future that there are many non-male users/moderators!
i just hi sir, because i want to answer from only your side, not other male moderators, anyways i will remember this thing in future.
2. Try the Atacama tutorial and just skip to the dada2 denoise-paired step.
i will try and shared the result which i will get
but sir in the Atacama tutorial, the command asking for barcode information since i mention in my earlier post that i dont have the barcode sequence and primer information (not provided by service company) how to use this tutorial without completed this command. image attached.
okay sir i got it your point. after the following the command of this tutorial i got 3167 feature with is quite low, and also when i saw the sequence stats it showed that 75-80 % reads are merged so for the improving these stats i am asking to you for some other tutorials.
No problem, thanks for understanding. If you ever want to draw the attention of a specific member on the forum you can call them by ‘atting’ them with @[their_name] . But for general questions like this I would recommend not calling specific individuals but rather make the question broad to the whole community.
Glad you got past your first issue, sounds like you are running into a second -and-unrelated- issue. Could you please start a new topic with the following details so that we can help you further there:
What is your sample type (soil, mouse species, human oral etc.)
How were these reads sequenced? (ex. Illumina 2x300 run)
What region (ex. V4? V3-V4, ITS etc)
Why do you think 3167 features is quite low? What do you expect?
Post the output of dada2-denoise stats visualization (denoising-stats.qzv if you’re following the Atacam tutorial)