# MetaPhlan3 --unknown\_estimation
**URL:** https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002
**Category:** MetaPhlAn
**Created:** [September 11, 2020, 11:57am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002 "2020-09-11T11:57:38Z")
**Posts on this page:** 20
**Page:** 1
### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 11, 2020, 11:57am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/1 "2020-09-11T11:57:38Z")
[metaphlan3outputEX.txt](https://forum.biobakery.org/uploads/short-url/xYYRXXM3qOrqB1iS4AkEwXUGQwH.txt) (73.4 KB)
Dear MetaPhlan3 developers,
I am very excited about this new release with so many more references 🙂
I did a testrun as this:
` for i in *.fastq; do metaphlan $i --input_type fastq --nproc 20 --unknown_estimation --index latest --add_viruses > ../metaphlan3/${i%.fastq}_profile.txt; done`
I am a bit confused about the output. Please check attached file. Why are there two rows named ‘UNKNOWN’? One has only ‘0’ but the first has very high values (80-90+ %) which seems a lot taken into account that this is human fecal samples.
When I sum up all relative abundances I end up with 150-180% which is strangem too.
Please help me interpret my results!
Thank you!
Stef
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 14, 2020, 8:59am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/2 "2020-09-14T08:59:53Z")
Hi Stef,
For fecal samples, it is a quite high value, is it possible that the sample contains contaminants like human sequences?
About the \>100% sum, the UNKNOWN value is referred to the sum of the relative abundances at one clade level, so if you sum up all the species’ relative abundance and add the UNKNOWN value you’ll get 100%.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 14, 2020, 1:17pm UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/3 "2020-09-14T13:17:53Z")
I have removed human reads before running metaphlan3 (which were 4% as the highest in one sample). So that should not be it. And why two rows with ‘UNKNOWN’ one being zero and the other above 80%? Could you please take a look at the output I posted?
I very much appreciate your help interpreting the results!
/Stef
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 14, 2020, 3:51pm UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/4 "2020-09-14T15:51:21Z")
That’s pretty strange, can you upload here all the bowtie2out files MetaPhlAn generated?
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 15, 2020, 6:23am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/5 "2020-09-15T06:23:22Z")
[PB.39.fastq.bowtie2out.txt](https://forum.biobakery.org/uploads/short-url/2kPXG4gxNSX1fL98rjU9BtKDGup.txt) (801.0 KB) [PB.41.fastq.bowtie2out.txt](https://forum.biobakery.org/uploads/short-url/yKsztmHwlL5GpJgTigJ5V1vnGIu.txt) (1.3 MB) [PB.42.fastq.bowtie2out.txt](https://forum.biobakery.org/uploads/short-url/hzrwdKC1eyFrqdtCF29xFguYAJk.txt) (3.7 MB)
Here are the bowtie2 output files of three samples.
Thank you so much for helping!
/Stef
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 15, 2020, 8:01am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/6 "2020-09-15T08:01:29Z")
Hi Stef,
I cannot reproduce the same behaviour (two UNKNOWN rows) after merging the three outputs. Which version of MetaPhlAn are you using?
[merged.txt](https://forum.biobakery.org/uploads/short-url/A8V1ojAjSAizcGlebgiywUpYF8d.txt) (23.3 KB)
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 15, 2020, 8:36am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/7 "2020-09-15T08:36:26Z")
Metaphlan3
I have more sample (70) so the problem could be somewhere else? How can I identify a potentially problematic sample?
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 15, 2020, 8:37am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/8 "2020-09-15T08:37:36Z")
Still, the unknown should not be above 80% since it is fecal samples after removal of host reads.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 15, 2020, 8:43am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/9 "2020-09-15T08:43:47Z")
I identified the issue! The second row of UNKNOWN came from the negative control. It was 100 there and 0 for all samples. When merging all samples without the NC it looks fine.
But I am still VERY worried about the UNKNOWN in my samples being above 80%!
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 18, 2020, 6:15am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/10 "2020-09-18T06:15:18Z")
Hi again,
Do you have any suggestions on how I can increase the mapping to reduce the % UNKNOWN read?
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 18, 2020, 2:18pm UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/11 "2020-09-18T14:18:09Z")
I’ll resolve this issue, it seems that the string printed when no output is available and the one for the unknown estimation are slightly different.
About increasing the mappability, the metagenome size seems below average, are these MiSeq reads?
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 18, 2020, 2:55pm UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/12 "2020-09-18T14:55:11Z")
Exactly! MiSeq data, 2x300 bp, about 2 Mreads pairs per sample, sometimes only 1M. Is there any useful fine tuning for fewer but longer reads?
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 18, 2020, 4:07pm UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/13 "2020-09-18T16:07:07Z")
Given the particularly longer read length, I’d try to use MetaPhlAn with a local alignment, you can do this by running MetaPhlAn with the `--bt2_ps sensitive-local` or `--bt2_ps very-sensitive-local` parameter.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 21, 2020, 6:13am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/15 "2020-09-21T06:13:37Z")
Thanks! I will!
Could you please tell me how exactly sensitive and very sensitive differ? I cannot find that information in the tutorial. And which min\_alignment\_len do you recommend?
/Stef
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 21, 2020, 8:37am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/16 "2020-09-21T08:37:28Z")
For the parameters definition, I’ll point you to the [Bowtie2 manual](http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml#bowtie2-options-sensitive) since it’s a bowtie2 parameter. I’d not decrease the min\_alignment\_len below 100, you should not have markers with that size and it should guarantee you to find enough hits.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 24, 2020, 6:25am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/17 "2020-09-24T06:25:28Z")
Hi Francesco,
Using the local alignment I could decrease the UNKNOWN by around half. So this is much better but still about 40% left as unknown. Do you have any further suggestions on how to optimise the parameters to longer MiSeq reads and shallow datasets?
Thank you!
Stef
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 24, 2020, 7:51am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/18 "2020-09-24T07:51:43Z")
I’m glad it worked out. 40% is a reasonable number for UNKWNOWN.
For longer reads, the tuneable parameters are the two you used before (`min_alignment_len` and `--bt2_ps`, and are the one that would mostly impact on the increase of mappability.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 24, 2020, 7:53am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/19 "2020-09-24T07:53:25Z")
Thank you for your help! So 40% is what you expect in fecal samples? Is there still so much dark matter?
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### Author:  [@fbeghini](https://forum.biobakery.org/u/fbeghini)
#### Post date: [September 24, 2020, 8:34am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/20 "2020-09-24T08:34:22Z")
Yes, the average mappability in stool samples is around 60%, I’ll point you to Figure 2A ([https://www.sciencedirect.com/science/article/pii/S0092867419300017#fig2](https://www.sciencedirect.com/science/article/pii/S0092867419300017#fig2)) from the Pasolli et al 2019 paper.
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### Author:  [@Stef](https://forum.biobakery.org/u/Stef)
#### Post date: [September 24, 2020, 8:36am UTC](https://forum.biobakery.org/t/metaphlan3-unknown-estimation/1002/21 "2020-09-24T08:36:59Z")
Well, then that’s great! I guess I am now ready to analyse my taxonomic profiles. Thank you so much! 😃
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