For each patient, I only have two files, with one for forward and one for reverse. The files are stored in a folder called Yang / Yang2, and the file name is like:
Part of the content of the first file (AB2S74_01_L001_R1_001.fastq) is shown below:
My questions are listed below.
What is last piece of the first line (GGAGCTAC+GAGCCTTA)? Is it sample ID? Do I need to remove it for downstream analysis? How to remove it?
The length of amplicons is about 250 bp for either forward or reverse? Is there a way to check if they are overlapped? Also I am not sure how to decide how many bps at the beginning or end that are needed to be trimmed (13 in the tutorial) and the amplicon length retained (200 in the following example) after taking overlapping into consideration?
qiime dada2 denoise-paired --i-demultiplexed-seqs demux-paired-end.qza --p-trim-left-f 13 --p-trim-left-r 13 --p-trunc-len-f 200 --p-trunc-len-r 200 --o-table table.qza --o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza
- I used Casava 1.8 to merge the paired-end files for each patient and followed the tutorial step by step to do downstream analysis. When I approached the step for alpha and beta diversity analysis, I was unable to continue using the following command.
qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table table.qza --p-sampling-depth 35000 --m-metadata-file sample-metadata.tsv --output-dir core-metrics-results
I tried to delete “–m-metadata-file sample-metadata.tsv”, but QIIME2 displayed the following message.
Error: Missing option: --m-metadata-file.
So do I need to create a metadata.tsv file? Since I used Casava 1.8 to import the data, what kind of file is needed for this option?
Look forward to your reply. Many thanks in advance!