Does a metadata file have to be provided to create a biom file, for subsequent analysis of beta diversity in Phinch?

[Erica_Dasi]

Greetings,

I am currently using the Galaxy Europe platform to analyze 16S sequencing data, while using the 16S Microbial Analysis tutorial (https://galaxyproject.github.io/training-material/topics/metagenomics/tutorials/mothur-miseq-sop/tutorial.html#otu-clustering). I have reached the step where the shared output file is combined with the mouse metadata file (i.e., mouse.dpw.metadata) to create a biom file, which is subsequently used in Phinch to examine beta diversity.

From my reading, it appears that the metadata provides additional technical details about the samples (e.g., day of collection). This information can be included with the output data generated in Galaxy to form the biom file. I modified the mouse.dpw.metadata to include my samples and the days that they were collected. However, I received an error when I used the make.biom tool and included this modified file.

I was able to proceed without using the metadata file. However, I was wondering how the Phinch program takes the metadata into account? For instance, the taxonomic bar chart that is produced in Phinch does not include information on the sampling days. As a result, I’m wanted to reach out to gain a better understanding of the purpose of the metadata file and whether it is important to include in the 16S sequencing analysis.

Thank you for your time and I look forward to hearing from you. Have a wonderful day!

Sincerely,
Erica

[jennaj]

Welcome @Erica_Dasi

I’m wanted to reach out to gain a better understanding of the purpose of the metadata file and whether it is important to include in the 16S sequencing analysis.

The answer is probably yes, otherwise you won’t be able to filter down to specific groups of samples with Mothur tools or later Phinch itself.

Where is “metadata” used?

The metadata input sets allow you to filter using the “groups - Groups to include” option on the Make.biom tool form.

The parsed metadata information is also then included in the final biom dataset output later input to Phinch, where it can be utilized/filtered on when using that tool.

Why is your metadata file failing?

There is probably a format problem. Extra spaces, etc can cause problems. The data should be tab-delimited, each column’s label/data needs to be formatted correctly, and the file should not contain any extra lines. The header also needs to be correctly formatted. How to check for format problems like this are similar to the help in this post.

Barcode Splitter Bug

It is very important that there is no extra “whitespace” anywhere in the file, meaning no extra spaces, no extra tabs, no extra trailing blank lines, and no other hidden characters (soft returns that Excel can introduce, and the like) on any line. The tool is picky about inputs.

I would suggest downloading your barcode dataset from Galaxy and examining it to check for extra whitespace. Using the “eye” (view dataset) icon will display the data in Galaxy but sometimes extra whitespace is not easy to detect that way.

More help:

• Tool manual that has an example “metadata” file and explains how it is used: https://www.mothur.org/wiki/Make.biom

• What this tutorial’s “metadata” file looks like. Source: https://zenodo.org/record/800651/files/mouse.dpw.metadata

  group dpw
  F3D0 0
  F3D1 1
  F3D141 141
  F3D142 142
  F3D143 143
  F3D144 144
  F3D145 145
  F3D146 146
  F3D147 147
  F3D148 148
  F3D149 149
  F3D150 150
  F3D2 2
  F3D3 3
  F3D5 5
  F3D6 6
  F3D7 7
  F3D8 8
  F3D9 9

• Example of a biom datasets, including those that incorporate metadata inputs. See: http://biom-format.org/documentation/format_versions/biom-2.1.html#example-biom-files

• Help for using Phinch: http://phinch.org/How-to-Get-Started && http://phinch.org/Tutorials