Hello!
I hope you guys are doing fine under the circumstances of the COVID19 emergency and thanks in advance for you time!
I think I am having trouble understanding the workflow of qiime and how it related to my specific project. My project consisted in taking gut samples (16S) from different individuals of the same species of sea urchin across different locations. I want to first know more about the taxonomic contents of the gut, including alpha, beta, phylogeny, and what ever other data I can squeeze out of it. Eventually once I acquire another data set (after sequencing) I want to add cytochrome B data from the individual sea urchins and correlate it with the data from the gut and metadata (location, etc.).
The data I received from the sequencing facility (16s) was generated by QIIME 1.9.1 :
sea urchin 16s qiime data folder
I have Qiime installed on a windows virtual box with the following specs:
The workflow I'm following is close to the Moving Pictures:
0. Unzip, Add meta data to BIOM Table (BIOMV210Format) generated by QIIME 1.9.1
(...in folder core-2762 in the data folder linked above)
biom add-metadata -i table_mc2762.biom -o seaurchintable.biom --observation-metadata-fp SeaUrchinMetadataQiime2.tsv
Output: seaurchintable.biom
1. Import BIOM Table into qiime2
qiime tools import
--input-path seaurchintable.biom
--type 'FeatureTable[Frequency]'
--input-format BIOMV210Format
--output-path seaurchinfeaturetable.qza
Output: seaurchinfeaturetable.qza
2. Import Phylogenetic Trees
qiime tools import
--input-path gg_13_8_otus/trees/97_otus.tree
--output-path unrooted-tree.qza
--type 'Phylogeny[Unrooted]'
Output: unrooted-tree.qza
3. Import Per-feature unaligned Sequence Data
qiime tools import
--input-path gg_13_8_otus/rep_set/97_otus.fasta
--output-path sequences.qza --type 'FeatureData[Sequence]'
4. Feature Table & Feature Data
qiime feature-table summarize
--i-table seaurchinfeaturetable.qza
--o-visualization seaurchinfeaturetable.qzv
--m-sample-metadata-file SeaUrchinMetadataQiime2.tsv
qiime feature-table tabulate-seqs
--i-data sequences.qza
--o-visualization sequences.qzv
5. Phylogenetic Diversity Tree
qiime phylogeny align-to-tree-mafft-fasttree
--i-sequences sequences.qza
--o-alignment aligned-rep-seqs.qza
--o-masked-alignment masked-aligned-rep-seqs.qza
--o-tree unrooted-tree.qza
--o-rooted-tree rooted-tree.qza
Output: Error
Command '['mafft', '--preservecase', '--inputorder', '--thread', '1', '/tmp/qiime2-archive-2voi5koo/1e445fb7-4cc7-45d7-a804-b160f18b1e32/data/dna-sequences.fasta']' returned non-zero exit status 1.
Upon searching the forum I came across these topics which may give me a possible way of tackling the problem. I want to make sure that I have the correct workflow up until now and make sure that i understand where I am going with this hence why i am writing this topic.
"mafft -> insufficient memory on the machine you are running this command on."
another insufficient memory error
or maybe that I have imported the files wrong...
hints that i can import as FeatureTable[Frequency] & FeatureData[Taxonomy]
or maybe
that i needed filtering the sequences...but i am unsure how to proceed with this and find the p-minus values according to the filtering tutorial.
Thanks in advance!
Also i used to run a discourse app back in the day you can add some pretty nice features like github integration etc. if you know how to do it without breaking it. let me know if you guys ever need any help.