# Rarefying raw file for taxonomic classification MetaPhlAn3

**URL:** <https://forum.biobakery.org/t/rarefying-raw-file-for-taxonomic-classification-metaphlan3/2083>\
**Category:** MetaPhlAn\
**Created:** [May 17, 2021, 8:14am UTC](https://forum.biobakery.org/t/rarefying-raw-file-for-taxonomic-classification-metaphlan3/2083 "2021-05-17T08:14:13Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![saras22](https://avatars.discourse-cdn.com/v4/letter/s/59ef9b/32.png) [@saras22](https://forum.biobakery.org/u/saras22)\
**Post date:** [May 17, 2021, 8:14am UTC](https://forum.biobakery.org/t/rarefying-raw-file-for-taxonomic-classification-metaphlan3/2083/1 "2021-05-17T08:14:13Z")

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Hi @fbeghini sir  
I have done taxonomic profiling using MetaPhlAn3 but my question is that my input files or raw files (.fastq.gz format) are of varying sizes ranging from 200 MB to 18GB.

- rarefy raw files?  
so do you think I need to rarefy my data before doing the taxonomic profiling? if yes then what is the method to do so?  
I am asking this question because after doing the analysis I got different results for different sizes of files. For e.g If the file was having 641 M Bases of sequencing yield then it gave me 62 species and when the file was having 4473 M bases sequencing yield I got 181 species for that particular file/sample.

- get the datasheet for top 100 or top 25 species  
I apologise for asking question related to other topic but I need to know when we use the flag --ftop 100 or --ftop n in merged\_profile.txt file we get n number of species or whatever level(phylum/class…etc) while making the heat map using #hclust2, how is it done what is the method? Do you take average of all the samples? Can I get the datasheet or a table for the top 25 features/clades?

Thanks a ton in Advance  
Saraswati

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**Author:** ![saras22](https://avatars.discourse-cdn.com/v4/letter/s/59ef9b/32.png) [@saras22](https://forum.biobakery.org/u/saras22)\
**Post date:** [July 2, 2021, 7:32am UTC](https://forum.biobakery.org/t/rarefying-raw-file-for-taxonomic-classification-metaphlan3/2083/2 "2021-07-02T07:32:40Z")

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Hi @fbeghini  
Please reply to the 1st question if you find any time because I found the answer to the 2nd question. Thanks.

Saraswati

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**Author:** ![alexis\_saldivar](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/alexis_saldivar/32/897_2.png) [@alexis\_saldivar](https://forum.biobakery.org/u/alexis_saldivar)\
**Post date:** [July 3, 2021, 8:28pm UTC](https://forum.biobakery.org/t/rarefying-raw-file-for-taxonomic-classification-metaphlan3/2083/3 "2021-07-03T20:28:35Z")

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If your samples come from different environments, then differences in diversity are to be expected. If they do not and you expect them to have a similar number of species, then this is probably caused by biases introduced by different sequencing depths, therefore it is unlikely that rarefying could improve your results.