Hello, I have some questions about starting with PE sequencing data. I have sequencing data. They are 16S MiSeq using EMP primers and barcodes. I follow the instructions from this tutorials (https://docs.qiime2.org/2019.10/tutorials/atacama-soils/)
I have some questions:
1> The first step, I need to run “qiime tools import” to generate a qza file (emp-paired-end-sequences.qza). It worked for me.
My questions here: It seems the fold that I imported must have three files and the name must be exactly forward.fastq.gz, reverse.fastq.gz, bacodes.fastq.gz. If I don’t use these file names. qiime2 give me errors?
So, everytime before I import my data, I have to rename those files?
2>Next, I need to run demuliplex command. This is an example:
qiime demux emp-paired
a> At the beginning, I suppose that I don’t need “–p-rev-comp-mapping-barcodes”. However, if I don’t add this, the scripts give me errors. The error is “No sequences were mapped to samples. Check that your barcodes are in the correct orientation (see the rev_comp_barcodes and/or rev_comp_mapping_barcodes options).”
What is the difference between rev_comp_barcodes and rev_comp_mapping_barcodes? In my case, only rev_comp_mapping_barcodes work. When should I use rev_comp_barcodes?
b>To be honest, I don’t know why I need to use reverse compliment. These data are my old data. I used to use QIIME 1 to split the library which is split_libraries_fastq.py. I don’t think QIIME1 does automatic reverser compliment for me. I just using the same column of barcode as I used in QIIME2. I don’t know why it didn’t work in QIIME2 (has to reverse compliment?)
c>This questions follow up with the above questions. After demulitplex, I compare the reads between Qiime1 and qiime 2 results. I found I lost 95% of reads using Qiime 2 demulitplex workflow?
I am not sure if I did it correctly or not. My barode is 12nt. I found a parameter-- “–p-golay-error-correction / --p-no-golay-error-correction Perform 12nt Golay error correction on the barcode reads.”
How many nt of barcode would QIIME2 think default? 8nt? Should I try both 12nt with and without reverse compliment parameters?
I lost too many reads.
d>Once I finish demultiplex, I created a qzv file, so I can visualize it. It requires running "qiime tools view " and open firefox to see the statistical results of demulitiplex.
I am using remote connection to a super computer? Is it possbile I can generate a txt file? this is not convenient to me.