I am requesting help in a command usage
So, my reads were already demultiplexed when I received them from the sequencing center. This is what they wrote after I asked them about quality filtering steps:
Reply: <<Cluster filtering and base calling was performed with Miseq control software v. 3.1 and real time analysis software (RTA) v 1.18.54 Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads >>
A sample read is named as follows:
The Quality offset reported in my results is 33.
So, here’s how I imported the sequence reads:
-created a manifest file named YIK-manifest (3 columns: sample-id (I supplied my own custom id), absolute filepath (the $path/name-of-read), direction (forward or reverse).
-ran the following command:
qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path YIK-manifest --output-path YIK-paired-end-demux.qza --input-format PairedEndFastqManifestPhred33
Is this correct?
2 more (small) questions:
a. The sequencing center says they did not use Cassava demultiplexed format, however, the above command taken from the qiime2 tutorial page is for Cassava paired-end demultiplexed format (https://docs.qiime2.org/2018.11/tutorials/importing/). I referred to this command because my reads are demultiplexed and paired-end reads. Instead of renaming name, I simply assigned them to sample-ids and did everything as in the Cassava format. Am I missing anything here? Did i do anything wrong (Note: I got a demux.qza output,…all the way to a taxonomy file, but just checking). Here is a screenshot of my manifest file:
b. What is the difference between phred33 and phred33V2? My command gives error message when I write phred33V2. The sequencing center said that they used MiSeq Reagent Kit v2 paired-end 250 (2 x 250bp). Is the phred33V2 linked to the v2 paired-end phrase? If so, why is it not working.
I know it’s a lot of questions, I am very sorry for taking up everyone’s time here! Would really appreciate if I can get a light on this! Thanks a ton.