Answers.Body: <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>
  
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Answers.UserDisplayName: Lei ZHANG
Answers.Body: <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>
  
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Answers.Text: The contaminants are probably numerous and/or unknown in a metagenome?
Answers.UserDisplayName: Sam Nicholls
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Answers.Text: 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.
Answers.UserDisplayName: GenoMax
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Answers.Text: 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`.
Answers.UserDisplayName: Sam Nicholls
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Answers.Text: @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
Answers.UserDisplayName: Chris_Rands
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Answers.UserDisplayName: GenoMax
Answers.Body: <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>
  
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Answers.LastActivityDate: 2017-06-07T10:45:03.643Z
Answers.LastEditDate: 2017-06-07T10:45:03.643Z
Answers.UserDisplayName: gringer
Body: <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>
  
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-06-07T07:55:44.743Z
Comments.Text: 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
Comments.UserDisplayName: Chris_Rands
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-06-07T08:06:08.987Z
Comments.Text: If it includes eukaryotes, it's probably not 16s. I guess I would assume shotgun metagenomic data.
Comments.UserDisplayName: gringer
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-06-07T10:24:16.270Z
Comments.Text: 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
Comments.UserDisplayName: Chris_Rands
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-06-07T10:46:01.773Z
Comments.Text: 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.
Comments.UserDisplayName: terdon
Tags: <read-mapping><metagenome>
Title: Accidental mapping of eukaryotic reads in a metagenomic dataset
ThreadText: # 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**: @username_5 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.
  
  
  ---
  
  