Metadata file path doesn't exist

Technical Support
1

Hello, I am analysing 16srrna from feces of pigs. V3-V4 region sequence were retrieved from the Sequencing company. Only Forward and Reverse sequences were provided. From that data, I import into qiime by the following command:

qiime tools import
--type 'SampleData[PairedEndSequencesWithQuality]'
--input-path MuPlus-manifest.tsv
--output-path paired-end-demux.qza
--input-format PairedEndFastqManifestPhred33V2
then i can follow the moving picture tutorial until reach Alpha and beta diversity analysis
with the following command:

qiime diversity core-metrics-phylogenetic
--i-phylogeny phylogeny-align-to-tree-mafft-fasttree/rooted_tree.qza
--i-table table.qza
--p-sampling-depth 5900
--m-metadata-file sample-metadata1.tsv
--output-dir diversity-core-metrics-phylogenetic

however, I got the following error massage:

"There was an issue with loading the file sample-metadata1.tsv as metadata:

Metadata file path doesn't exist, or the path points to something other than a file. Please check that the path exists, has read permissions, and points to a regular file (not a directory): sample-metadata1.tsv

There may be more errors present in the metadata file. To get a full report, sample/feature metadata files can be validated with Keemei: https://keemei.qiime2.org

Find details on QIIME 2 metadata requirements here: Metadata in QIIME 2 — QIIME 2 2021.4.0 documentation"

sample-metadata1.tsv file I used consist only sample-id column. This file locate in the right directory and checked with Keemei.

How can I fix this promblem and go on?

Best Regards

2

Hello @Sarthorn,

First, welcome to the :qiime2: forum!

From a very practical perspective, make sure that when you run the command, your metadata file is in the folder you're currently working out of. Take the steps listed in the error message, and make sure that you: (1) have a file int hat location (and not say a folder) and (2) the file is readable. The rest warns you about other potential problems.
You may also have to try passing an additional column in your metadata that has some value, I haven't actually experimented with emperor visualization when no metadata beyond IDs are provided.

I think a more important question (although possibly slightly ruder) is where your metadata is. Like, if you spent money to sequence samples, you likely had a research question and a hypothesis about what you wanted to test. Im going to assume it wasn't just "what does pig poo look like :pig2:?" and instead related to something about species, diet, location, treatment, age, sex, or something else important and interesting. You probably also have some confounders you might want to think about (pen, if pigs are coprophagic; age, sex, whatever). Before you move forward, my recommendation would be to stop, collate this information, and make sure it's put together before you move forward.

Best,
Justine

3

Thank you, Justine. After I try to add column describe their groups. Its work.

Thank you.