6. Taxonomic investigation

6.1. Preface

We want to investate if there are sequences of other species in our collection of sequenced DNA pieces. We hope that most of them are from our species that we try to study, i.e. the DNA that we have extracted and amplified. This might be a way of quality control, e.g. have the samples been contaminated? Lets investigate if we find sequences from other species in our sequence set.

We will use the tool Kraken to assign taxonomic classifications to our sequence reads. Let us see if we can id some sequences from other species.

Note

You will encounter some To-do sections at times. Write the solutions and answers into a text-file.

6.2. Overview

The part of the workflow we will work on in this section can be viewed in Fig. 6.1.

../_images/workflow5.png

Fig. 6.1 The part of the workflow we will work on in this section marked in red.

6.3. Kraken

We will be using a tool called Kraken [WOOD2014]. This tool uses k-mers to assign a taxonomic labels in form of NCBI Taxonomy to the sequence (if possible). The taxonomic label is assigned based on similar k-mer content of the sequence in question to the k-mer content of reference genome sequence. The result is a classification of the sequence in question to the most likely taxonomic label. If the k-mer content is not similar to any genomic sequence in the database used, it will not assign any taxonomic label.

6.4. Before we start

Lets see how our directory structure looks so far:

cd ~/analysis
ls -1F
assembly/
data/
mappings/
SolexaQA/
SolexaQA++
trimmed/
trimmed-fastqc/
trimmed-solexaqa/

6.5. Installation

Use conda in the same fashion as before to install Kraken:

source activate ngs
conda install kraken-all

Now we create a directory where we are going to do the analysis and we will change into that directory too.

# make sure you are in your analysis root folder
cd ~/analysis

# create dir
mkdir kraken
cd kraken

Now we need to create or download a Kraken database that can be used to assign the taxonomic labels to sequences. We opt for downloading the pre-build "minikraken" database from the Kraken website:

curl -O https://ccb.jhu.edu/software/kraken/dl/minikraken.tgz

# alternatively we can use wget
wget https://ccb.jhu.edu/software/kraken/dl/minikraken.tgz

# once the download is finished, we need to extract the archive content
# it will create a directory: "minikraken_20141208/"
tar -xvzf minikraken.tgz

Attention

Should the download fail. Please find links to alternative locations on the Downloads page.

Note

The "minikraken" database was created from bacteria, viral and archaea sequences. What are the implications for us when we are trying to classify our sequences?

6.6. Usage

Now that we have installed Kraken and downloaded and extracted the minikraken database, we can attempt to investigate the sequences we got back from the sequencing provider for other species as the one it should contain. We call the Kraken tool and specify the database and fasta-file with the sequences it should use. The general command structure looks like this:

kraken --only-classified-output --db minikraken_20141208 example.fa > example.kraken

However, we may have fastq-files, so we need to use --fastq-input which tells Kraken that it is dealing with fastq-formated files. Here, we are investigating one of the unmapped paired-end read files of the evolved line.

kraken --only-classified-output --db minikraken_20141208 --fastq-input ../mappings/evolved-6.sorted.unmapped.R1.fastq > evolved-6-R1.kraken

Note

We are for now only interested in the portion of sequeuces that can be classified, that is why we supplied the option --only-classified-output to the Kraken command.

This classification may take a while, depending on how many sequences we are going to classify. The resulting content of the file "evolved-6-R1.kraken" looks similar to the following example:

C       M02810:197:000000000-AV55U:1:1101:10078:18384/1 2157    151     0:99 2157:1 0:1 2157:1 0:19
C       M02810:197:000000000-AV55U:1:1101:10573:27304/1 364745  150     0:43 364745:1 0:76
C       M02810:197:000000000-AV55U:1:1101:10852:5722/1  37665   151     0:33 37665:1 0:87
C       M02810:197:000000000-AV55U:1:1101:11429:10330/1 374840  101     0:17 374840:1 0:4 374840:1 0:2 374840:1 0:45
C       M02810:197:000000000-AV55U:1:1101:11705:24355/1 2157    151     0:1 2157:1 0:119

Each sequence classified by Kraken results in a single line of output. Output lines contain five tab-delimited fields; from left to right, they are:

  1. C/U: one letter code indicating that the sequence was either classified or unclassified.
  2. The sequence ID, obtained from the FASTA/FASTQ header.
  3. The taxonomy ID Kraken used to label the sequence; this is 0 if the sequence is unclassified and otherwise should be the NCBI Taxonomy identifier.
  4. The length of the sequence in bp.
  5. A space-delimited list indicating the lowest common ancestor (in the taxonomic tree) mapping of each k-mer in the sequence. For example, 562:13 561:4 A:31 0:1 562:3 would indicate that:
    • the first 13 k-mers mapped to taxonomy ID #562
    • the next 4 k-mers mapped to taxonomy ID #561
    • the next 31 k-mers contained an ambiguous nucleotide
    • the next k-mer was not in the database
    • the last 3 k-mers mapped to taxonomy ID #562

Note

The Kraken manual can be accessed here.

6.7. Investigate taxa

We can use the webpage NCBI TaxIdentifier to quickly get the names to the taxonomy identifier. However, this is impractical as we are dealing potentially with many sequences. Kraken has some scripts that help us understand our results better.

6.7.1. kraken-report

First, we generate a sample-wide report of all taxa found. This can be achieved with the tool kranken-report.

kraken-report --db minikraken_20141208 evolved-6-R1.kraken > evolved-6-R1.kraken.report

The first few lines of an example report are shown below.

  0.00  0       0       U       0       unclassified
100.00  2665    47      -       1       root
 54.56  1454    0       -       131567    cellular organisms
 38.50  1026    1023    D       2157        Archaea
  0.08  2       0       P       651137        Thaumarchaeota
  0.08  2       0       -       651142          unclassified Thaumarchaeota
  0.08  2       0       G       1048752           Candidatus Caldiarchaeum
  0.08  2       2       S       311458              Candidatus Caldiarchaeum subterraneum
  0.04  1       0       P       28890         Euryarchaeota
  0.04  1       0       C       183967          Thermoplasmata

The output of kraken-report is tab-delimited, with one line per taxon. The fields of the output, from left-to-right, are as follows:

  1. Percentage of reads covered by the clade rooted at this taxon
  2. Number of reads covered by the clade rooted at this taxon
  3. Number of reads assigned directly to this taxon
  4. A rank code, indicating (U)nclassified, (D)omain, (K)ingdom, (P)hylum, (C)lass, (O)rder, (F)amily, (G)enus, or (S)pecies. All other ranks are simply "-".
  5. NCBI Taxonomy ID
  6. indented scientific name

Note

If you want to compare the taxa content of different samples to another, one can create a report whose structure is always the same for all samples, disregarding which taxa are found (obviously the percentages and numbers will be different).

We can cerate such a report using the option --show-zeros which will print out all taxa (instead of only those found). We then sort the taxa according to taxa-ids (column 5), e.g. sort -n -k5.

kraken-report *--show-zeros* --db minikraken_20141208 evolved-6-R1.kraken | **sort -n -k5** > evolved-6-R1.kraken.report.sorted

The report is not ordered according to taxa ids and contains all taxa in the database, even if they have not been found in our sample and are thus zero. The columns are the same as in the former report, however, we have more rows and they are now differently sorted, according to the NCBI Taxonomy id.

6.7.2. kraken-translate

For every sequence in our sample and its predicted taxonomic identifier, we can attach the taxonomic names with kraken-translate.

kraken-translate --mpa-format --db minikraken_20141208 evolved-6-R1.kraken > evolved-6-R1.kraken.names

An example output looks like this:

M02810:197:000000000-AV55U:1:1101:10078:18384/1 d__Archaea
M02810:197:000000000-AV55U:1:1101:10573:27304/1 d__Viruses|f__Baculoviridae|g__Betabaculovirus|s__Choristoneura_occidentalis_granulovirus
M02810:197:000000000-AV55U:1:1101:10852:5722/1  d__Viruses|f__Phycodnaviridae|g__Phaeovirus|s__Ectocarpus_siliculosus_virus_1
M02810:197:000000000-AV55U:1:1101:11429:10330/1 d__Viruses|f__Microviridae|g__Microvirus|s__Enterobacteria_phage_phiX174_sensu_lato
M02810:197:000000000-AV55U:1:1101:11705:24355/1 d__Archaea
M02810:197:000000000-AV55U:1:1101:12206:13333/1 d__Viruses|o__Herpesvirales|f__Alloherpesviridae|g__Cyprinivirus|s__Cyprinid_herpesvirus_3
M02810:197:000000000-AV55U:1:1101:12336:11626/1 d__Viruses|f__Baculoviridae|g__Betabaculovirus|s__Choristoneura_occidentalis_granulovirus
M02810:197:000000000-AV55U:1:1101:12707:5809/1  d__Archaea
M02810:197:000000000-AV55U:1:1101:12741:21685/1 d__Viruses|f__Baculoviridae|g__Betabaculovirus|s__Choristoneura_occidentalis_granulovirus
M02810:197:000000000-AV55U:1:1101:12767:27821/1 d__Archaea

Here, each sequence that got classified is present in one row. The nomenclature for the names is preceded with an letter according to its rank, e.g. (d)omain, (k)ingdom, (p)hylum, (c)lass, (o)rder, (f)amily (g)enus, or (s)pecies. Taxonomy assignments above the superkingdom (d) rank are represented as just root.

6.7.3. Visualisation

We use the Krona tools to create a nice interactive visualisation of the taxa content of our sample [ONDOV2011]. Fig. 6.2 shows an example (albeit an artificial one) snapshot of the visualisation Krona provides. Fig. 6.2 is a snapshot of the interactive web-page similar to the one we try to create.

../_images/krona.png

Fig. 6.2 Example of an Krona output webpage.

Install Krona with:

source activate ngs
conda install krona

First some house-keeping to make the Krona installation work. Do not worry to much about what is happening here.

# we delete a symbolic link that is not correct
rm -rf ~/miniconda3/envs/ngs/opt/krona/taxonomy

# we create a directory in our home where the krona database will live
mkdir -p ~/krona/taxonomy

# now we make a symbolic link to that directory
ln -s ~/krona/taxonomy ~/miniconda3/envs/ngs/opt/krona/taxonomy

# now we copy some scripts around, this is necessary as krona installation fails to do this
cp -r ~/miniconda3/envs/ngs/opt/krona/scripts ~/miniconda3/envs/py3-kraken/bin/

We need to build a taxonomy database for Krona. However, if this fails we will skip this step and just download a pre-build one. Lets first try to build one.

ktUpdateTaxonomy.sh ~/krona/taxonomy

Now, if this fails, we download a pre-build taxonomy database for krona.

# Download pre-build database
curl -O http://compbio.massey.ac.nz/data/taxonomy.tab.gz

# we unzip the file
gzip -d taxonomy.tab.gz

# we move the unzipped file to our taxonomy directory we specified in the step before.
mv taxonomy.tab ~/krona/taxonomy

Attention

Should this also fail we can download a pre-build database on the Downloads page via a browser.

Now we use the tool ktImportTaxonomy from the Krona tools to crate the html web-page:

cat evolved-6-R1.kraken | cut -f 2,3 > evolved-6-R1.kraken.krona
ktImportTaxonomy evolved-6-R1.kraken.krona
firefox taxonomy.krona.html

What happens here is that we extract the second and third column from the Kraken results. Afterwards, we input these to the Krona script, and open the resulting web-page in a bowser. Done!

References

[ONDOV2011]Ondov BD, Bergman NH, and Phillippy AM. Interactive metagenomic visualization in a Web browser. BMC Bioinformatics, 2011, 12(1):385.
[WOOD2014]Wood DE and Steven L Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biology, 2014, 15:R46. DOI: 10.1186/gb-2014-15-3-r46.