Metadate file/ phylogenetic tree

[Nasute (Julissa)]

Hello everyone do the sequencing of some amplicons RNA16s in illumina MiSeq, in the company macrogen and they sent me several files the problem is that I have not identified which is the file that I use to validate my metadata since my data correspond to amplicons of 4 samples in total, and I still do not know which is the file that I must validate in keemei, since I omit this step and now to be able to elaborate the phylogenetic tree for my diversity analysis

[tomasz (Tomasz Kościółek)]

Hi @Nasute!

If I understand you correctly you got several files from macrogen and you’re wondering:

1. which file to validate in Keemei?
2. what are those files you got from the company?

Keemei is a tool to validate QIIME (1 or 2) mapping files and this is the file that you prepare yourself. You can think of it as all information about your samples outside of the sequencing facility. More information about what we understand as mapping files in QIIME2 can be found here.

My guess would be that macrogen sent you demultiplexed (per-sample) sequencing files and a manifest explaining which file is what. If that is the case you don’t need to worry about demultiplexing step, so information about barcode sequences and linker primer sequences is not required - they already did that for you! All you need to do now is to import this data as a QIIME2 artifact. And this document can help you out.

Does this help?

Tomasz

[Nasute (Julissa)]

Dear Tomas

Exactly, import my files through a manifest file like the following:
sample-id,absolute-filepath,direction
sample-1,/home/julissa/Documentos/Contigs/RJ8-10_1.fastq,forward
sample-2,/home/julissa/Documentos/Contigs/SCI8-12_1.fastq,forward
sample-3,/home/julissa/Documentos/Contigs/T2EEM_1.fastq,forward
sample-4,/home/julissa/Documentos/Contigs/ZT5-6_1.fastq,forward
sample-1,/home/julissa/Documentos/Contigs/RJ8-10_2.fastq,reverse
sample-2,/home/julissa/Documentos/Contigs/SCI8-12_2.fastq,reverse
sample-3,/home/julissa/Documentos/Contigs/T2EEM_2.fastq,reverse
sample-4,/home/julissa/Documentos/Contigs/ZT5-6_2.fastq,reverse

Can I import these files to convert them into qiime artifacts through a manifest file?

What would be the next step in order to build a tree for phylogenetic diversity analyses?

thank you so much for your help

Julissa

[tomasz (Tomasz Kościółek)]

yes, you should be able to do exactly that by following the instructions here.

Once you have your sequences imported as a QIIME2 artifact you would need to (following the tutorial):

1. construct a feature table by denoising your sequences (with deblur or DADA2).
2. generate a phylogenetic tree by either:
   a. de novo tree construction (as in the tutorial linked above)
   b. using an insertion tree from q2-fragment-insertion plugin

Tomasz

[Nasute (Julissa)]

Thank you very much already running @tomasz