<p>You can use <a href="https://ccb.jhu.edu/software/centrifuge/" rel="nofollow noreferrer">centrifuge</a> with the NT library to profile the taxonomy, and then remove the reads from eukaryotes. </p>

CC BY-SA 3.0
2017-06-07T03:49:20.207Z
2017-06-07T03:49:20.207Z
Lei ZHANG
<p>If you know the eukaryotic contaminant present you could use <code>bbsplit.sh</code> from <a href="https://sourceforge.net/projects/bbmap/" rel="nofollow noreferrer">BBMap suite</a> to split/bin reads first using a reference for that contaminant (into one or as many bins as reference sequences you provide). </p>

CC BY-SA 3.0
2017-06-07T11:23:29.650Z
The contaminants are probably numerous and/or unknown in a metagenome?
Sam Nicholls
CC BY-SA 3.0
2017-06-07T11:27:20.717Z
My comment was with reference to the eukaryote being the contaminant. The original question refers to a vague "both eukaryotes and prokaryotes" specification. Hopefully there is a small number of known "contaminants". In absence of concrete information none of the tools/answers are going to be a perfect solution.
GenoMax
CC BY-SA 3.0
2017-06-07T11:32:14.443Z
Sure, I was just saying that typically in metagenomics, you will capture many eukaryotes (that you may deem contaminants) in your sample. Often these environments are lacking in good references, too. In scenarios such as this, we often have to rely on less effective solutions than `BBMap`.
Sam Nicholls
CC BY-SA 3.0
2017-06-07T11:59:42.347Z
@SamNicholls if the sample is from a body site (like the human gut) then there is only likely 1 major eukaryotic contaminant (i.e. human DNA; ignoring rare microbial eukaryotes or food), but if you're trawling the ocean or something will be a lot of eukaryotes; think the question needs clarifying
Chris_Rands
CC BY-SA 3.0
2017-06-06T21:32:30.483Z
2017-06-06T21:32:30.483Z
GenoMax
<p><em>Answer from <a href="https://www.reddit.com/user/Romanticon" rel="nofollow noreferrer">/u/Romanticon</a> on reddit. The original answer can be found <a href="https://www.reddit.com/r/bioinformatics/comments/6dfu3c/question_about_eggnog_mapper_and_annotation_in/di2qv4a/" rel="nofollow noreferrer">here</a></em></p>

<blockquote>
  <p>Depending on the length of your reads, the level of error in the
  reads, the quality of the database, and the specificity settings you
  give DIAMOND, you can change the level of "bleed" that you get - but
  you'll always have a couple reads, especially at metagenome dataset
  sizes, that will be annotated incorrectly.</p>
  
  <p>But you'll always have this happen, whenever you annotate any large
  dataset. The best way to handle it is to set a threshold after
  annotation that removes wrongly annotated reads (looking for 'clearly
  wrong' organisms in your annotated dataset can help you figure out
  where to set the threshold level - there's probably no Bos taurus
  reads in a mouse metagenome).</p>
  
  <p>You can do things like enable the sensitive flag on the DIAMOND
  annotation search to help improve annotation accuracy.</p>
</blockquote>

CC BY-SA 3.0
2017-06-06T13:31:44.300Z
2017-06-07T10:45:03.643Z
2017-06-07T10:45:03.643Z
gringer
<p><em>This is a question from <a href="https://www.reddit.com/user/wipeyourmit" rel="nofollow noreferrer">/u/wipeyourmit</a> on reddit. The original post can be found <a href="https://www.reddit.com/r/bioinformatics/comments/6dfu3c/question_about_eggnog_mapper_and_annotation_in/" rel="nofollow noreferrer">here</a>.</em></p>

<blockquote>
  <p>If I have a metagenomic dataset that contains reads from both
  eukaryotes and prokaryotes and then I annotate by running DIAMOND or
  HMMER against a bacterial database how much of a risk do I run of
  eukaryotic reads being annotated in the process?</p>
  
  <p>I was hoping to use the eggNOG mapper to search against the bacterial
  and archaeal databases and to exclude the eukaryotic portion of my
  dataset. Is the eukaryotic filtering something that I would need to do
  in a step prior to this?</p>
</blockquote>

CC BY-SA 3.0
2017-06-07T07:55:44.743Z
Can you be more specific; by metagenomic data, do you mean shotgun metagenomic data or 16S? I assume without depletion of host DNA or an enrichment protocol? What is the sample site and species? For example, the ratio of prokaryotic:eukaryotic reads is much higher in the human gut than most other human body sites
Chris_Rands
CC BY-SA 3.0
2017-06-07T08:06:08.987Z
If it includes eukaryotes, it's probably not 16s. I guess I would assume shotgun metagenomic data.
gringer
CC BY-SA 3.0
2017-06-07T10:24:16.270Z
I guess this is a disadvantage of cross-posting, one can't really clarify the original question. Note cross-posting is being discussed now on meta https://bioinformatics.meta.stackexchange.com/questions/78/please-stop-cross-posting-behavior
Chris_Rands
CC BY-SA 3.0
2017-06-07T10:46:01.773Z
Please don't do this. If you want to repeat a question, it must be one for which you can provide clarification. Otherwise, the exercise is futile.
terdon
<read-mapping><metagenome>
Accidental mapping of eukaryotic reads in a metagenomic dataset
# Accidental mapping of eukaryotic reads in a metagenomic dataset

(Asked by: username_0 on 2017-06-06)

*This is a question from [/u/wipeyourmit](https://www.reddit.com/user/wipeyourmit) on reddit. The original post can be found [here](https://www.reddit.com/r/bioinformatics/comments/6dfu3c/question_about_eggnog_mapper_and_annotation_in/).*

> If I have a metagenomic dataset that contains reads from both
> eukaryotes and prokaryotes and then I annotate by running DIAMOND or
> HMMER against a bacterial database how much of a risk do I run of
> eukaryotic reads being annotated in the process?
>
> I was hoping to use the eggNOG mapper to search against the bacterial
> and archaeal databases and to exclude the eukaryotic portion of my
> dataset. Is the eukaryotic filtering something that I would need to do
> in a step prior to this?

**Tags:** read-mapping, metagenome
> **Comments:**
> **username_1**: Can you be more specific; by metagenomic data, do you mean shotgun metagenomic data or 16S? I assume without depletion of host DNA or an enrichment protocol? What is the sample site and species? For example, the ratio of prokaryotic:eukaryotic reads is much higher in the human gut than most other human body sites  
> **username_0**: If it includes eukaryotes, it's probably not 16s. I guess I would assume shotgun metagenomic data.  
> **username_1**: I guess this is a disadvantage of cross-posting, one can't really clarify the original question. Note cross-posting is being discussed now on meta https://bioinformatics.meta.stackexchange.com/questions/78/please-stop-cross-posting-behavior  
> **username_2**: Please don't do this. If you want to repeat a question, it must be one for which you can provide clarification. Otherwise, the exercise is futile.  

---

## 3 answers

### Answer by username_3 on 2017-06-07. Score: 3

You can use [centrifuge](https://ccb.jhu.edu/software/centrifuge/) with the NT library to profile the taxonomy, and then remove the reads from eukaryotes.


---

### Answer by username_4 on 2017-06-06. Score: 3

If you know the eukaryotic contaminant present you could use `bbsplit.sh` from [BBMap suite](https://sourceforge.net/projects/bbmap/) to split/bin reads first using a reference for that contaminant (into one or as many bins as reference sequences you provide).

> **Comments:**
> **username_5**: The contaminants are probably numerous and/or unknown in a metagenome?  
> **username_4**: My comment was with reference to the eukaryote being the contaminant. The original question refers to a vague "both eukaryotes and prokaryotes" specification. Hopefully there is a small number of known "contaminants". In absence of concrete information none of the tools/answers are going to be a perfect solution.  
> **username_5**: Sure, I was just saying that typically in metagenomics, you will capture many eukaryotes (that you may deem contaminants) in your sample. Often these environments are lacking in good references, too. In scenarios such as this, we often have to rely on less effective solutions than `BBMap`.  
> **username_1**: @SamNicholls if the sample is from a body site (like the human gut) then there is only likely 1 major eukaryotic contaminant (i.e. human DNA; ignoring rare microbial eukaryotes or food), but if you're trawling the ocean or something will be a lot of eukaryotes; think the question needs clarifying  

---

### Answer by username_0 on 2017-06-06. Score: 1

*Answer from [/u/Romanticon](https://www.reddit.com/user/Romanticon) on reddit. The original answer can be found [here](https://www.reddit.com/r/bioinformatics/comments/6dfu3c/question_about_eggnog_mapper_and_annotation_in/di2qv4a/)*

> Depending on the length of your reads, the level of error in the
> reads, the quality of the database, and the specificity settings you
> give DIAMOND, you can change the level of "bleed" that you get - but
> you'll always have a couple reads, especially at metagenome dataset
> sizes, that will be annotated incorrectly.
>
> But you'll always have this happen, whenever you annotate any large
> dataset. The best way to handle it is to set a threshold after
> annotation that removes wrongly annotated reads (looking for 'clearly
> wrong' organisms in your annotated dataset can help you figure out
> where to set the threshold level - there's probably no Bos taurus
> reads in a mouse metagenome).
>
> You can do things like enable the sensitive flag on the DIAMOND
> annotation search to help improve annotation accuracy.


---

