I am new to bioinformatics and newer to QIIME. I have an excel file with sequenced/ demultiplexed data and I need to use QIIME to analyze the data bt I need to know what it actually does. does it tell me how my samples similar or different than the reference? or what?
Hi Heba, you would typically use QIIME if you have environmental samples where you want to assess microbial diversity across the samples. So, which clusters of distinct OTUs are here, which ones are there, and what was the turnover. For these types of analyses, the reference - if you can call it that - might be the database that you use for identifying the different clusters using BLAST searches.
This is really a broad question.
You should probably start reading up in literature what one is able to perform with qiime. A good start is actually to visit their website and read up on user documentation, where you can also find numerous examples of different types of analysis that cab be performed: https://docs.qiime2.org/2019.7/
I would suggest you few things:
1) If you are new to bioinfo/QIIME, please read a bit about the metagenomics, its purpose and metagenomics data processing with QIIME
2) If you have raw data, please use it and perform all the analysis in QIIME
3) I wonder why would you have all the data in excel? Use of excel, word or similar is not recommended while processing the data.
4) try to learn basic command line usage in Linux/Mac or Linux subsystem in windows 10, and use text processing programs like notepad++ or atom. Do not use word or excel.
Hi Heba Huessin ,
QIIME contains a set of tools to obtain microbial community composition from your 16s Metabarcoding sequence data.
The excel file with you (most probably) the result from QIIME.
Once you completed sequencing of your samples on any Next Generation Sequencing machine, you will receive DNA sequences either in FASTA or FASTQ format (according to your preference.)
QIIME takes these sequence file as input.
Your sequence file contain forward reads and reverse reads, Mostly named in the format of (XXX.R1.fastq or XXXR2.fastq )
The first step in analysing these sequences is
(1) merging the reads, you combine forward and reverse reads to obtain a single sequence file.
(2) next step is demultiplexing your sequences. For this purpose and many other following steps , you need a text file called 'mapping file' , which contains all the metadata of the sequences (eg:name of samples, barcodes used etc).
(3) once you demultiplexed your sequences, you have to pick OTUs from these sequences. (OTUs are a cluster of sequences aligned with a predefined similarity. generally 97% of similarity is used to cluster). OTUs can be considered as vague form of a species (but remember a single species can have multiple OTUs).
In QIIME you have different OTU picking strategies, choose the one best suitable for you (for eg: if your samples are originated from well characterised locations like gut microbiome, you can use closed reference OTU picking method).
(4)once OTU picking is completed through your prefered OTU picking strategy, the output file will be a BIOM file.
this BIOM file used for all the further analysis in QIIME (like statistical analysis and obtaining different types of graphical representation of your data.)
biom file can only be read by QIIME, to get the OTU table in human readable form you have to convert the biom file into tsv, (I think the excel file you have is the output from the biom file conversion ).
You can proceed to do statistics in QIIME or you can use any other platforms like R at this stage.
QIIME webpage have a complete guide for all the scripts used.
Hope this helps.
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