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# Taxonomic Assignment

## Overview

Teaching: 30 min
Exercises: 15 min
Questions
• How can I know to which taxa my sequences belong?

Objectives
• Understand how taxonomic assignment works.

• Use Kraken to assign taxonomies to reads and contigs.

• Visualize taxonomic assignations in graphics.

## What is taxonomic assignment?

Taxonomic assignment is the process of assigning an Operational Taxonomic Unit (OTUs, that is, groups of related individuals) to sequences, that can be reads or contigs. To assign an OTU to a sequence it is compared against a database, but this comparison can be done in different ways. The comparison database in this assignment process must be constructed using complete genomes. There are many programs for doing taxonomic mapping, almost all of them follows one of the next strategies:

1. BLAST: Using BLAST or DIAMOND, these mappers search for the most likely hit for each sequence within a database of genomes (i.e. mapping). This strategy is slow.

2. K-mers: A genome database is broken into pieces of length k, so as to be able to search for unique pieces by taxonomic group, from lowest common ancestor (LCA), passing through phylum to species. Then, the algorithm breaks the query sequence (reads, contigs) into pieces of length k, look for where these are placed within the tree and make the classification with the most probable position.
Figure 1. Lowest common ancestor assignment example.

3. Markers: They look for markers of a database made a priori in the sequences to be classified and assign the taxonomy depending on the hits obtained.

A key result when you do taxonomic assignment of metagenomes is the abundance of each taxa or OTU in your sample. The absolute abundance of a taxon is the number of sequences (reads or contigs, depending on what you did) assigned to it. And its relative abundance is the proportion of sequences assigned to it. It is important to be aware of the many biases that that can skew the abundances along the metagenomics workflow, shown in the figure, and that because of them we may not be obtaining the real abundance of the organisms in the sample.

Figure 2. Abundance biases during a metagenomics protocol.

## Using Kraken 2

Kraken 2 is the newest version of Kraken, a taxonomic classification system using exact k-mer matches to achieve high accuracy and fast classification speeds. kraken2 is already installed in the metagenome environment, lets have a look at kraken2 help.

$kraken2  Need to specify input filenames! Usage: kraken2 [options] <filename(s)> Options: --db NAME Name for Kraken 2 DB (default: none) --threads NUM Number of threads (default: 1) --quick Quick operation (use first hit or hits) --unclassified-out FILENAME Print unclassified sequences to filename --classified-out FILENAME Print classified sequences to filename --output FILENAME Print output to filename (default: stdout); "-" will suppress normal output --confidence FLOAT Confidence score threshold (default: 0.0); must be in [0, 1]. --minimum-base-quality NUM Minimum base quality used in classification (def: 0, only effective with FASTQ input). --report FILENAME Print a report with aggregrate counts/clade to file --use-mpa-style With --report, format report output like Kraken 1's kraken-mpa-report --report-zero-counts With --report, report counts for ALL taxa, even if counts are zero --report-minimizer-data With --report, report minimizer and distinct minimizer count information in addition to normal Kraken report --memory-mapping Avoids loading database into RAM --paired The filenames provided have paired-end reads --use-names Print scientific names instead of just taxids --gzip-compressed Input files are compressed with gzip --bzip2-compressed Input files are compressed with bzip2 --minimum-hit-groups NUM Minimum number of hit groups (overlapping k-mers sharing the same minimizer) needed to make a call (default: 2) --help Print this message --version Print version information If none of the *-compressed flags are specified, and the filename provided is a regular file, automatic format detection is attempted.  In addition to our input files we also need a database with which to compare them. There are several databases compatible to be used with kraken2 in the taxonomical assignment process. ## Very important to know your database! The database you use will determine the result you get for your data. Imagine you are searching for a lineage that was recently discovered and it is not part of the available databases. Would you find it? Minikraken is a popular database that attempts to conserve its sensitivity despite its small size (Needs 8GB of RAM for the assignment). Unfortunately although it is much smaller that most databases, it is not small enough to be run by the machines we are using, so we won’t be able to run kraken2. We can check our available RAM with free -hto be sure of this. $ free -h

              total        used        free      shared  buff/cache   available
Mem:           3.9G        272M        3.3G         48M        251M        3.3G
Swap:            0B          0B          0B


If we were to download the database we would use the following command:

$curl -O ftp://ftp.ccb.jhu.edu/pub/data/kraken2_dbs/old/minikraken2_v2_8GB_201904.tgz$ tar -xvzf minikraken2_v2_8GB_201904.tgz


## Exercise 1: Remembering commands

Fill in the blanks to decompress the following file minikraken2_v2_8GB_201904.tgz.

$____ -xvzf ____.tgz  ## Solution $ tar -xvzf  minikraken2_v2_8GB_201904.tgz


tar command is used in linux to decompress files, so in this case it would extract the content of the compressed file minikraken2_v2_8GB_201904.tgz

## Taxonomic assignment of metagenomic reads

As we have learned, taxonomic assignment can be attempted before the assembly process. In this case we would use FASTQ files as inputs, which would be JP4D_R1.trim.fastq.gz and JP4D_R2.trim.fastq.gz. And the outputs would be two files: the report JP4D.report and the kraken file JP4D.kraken.

To run kraken2 we would use a command like this:

$mkdir TAXONOMY_READS$ kraken2 --db kraken-db --threads 8 --paired --fastq-input JP4D_R1.trim.fastq.gz JP4D_R2.trim.fastq.gz --o


Since we can’t run kraken2 here, we precomputed its results in a server, i.e. a more powerful machine. In the server we ran kraken2 and obtainedJP4D-kraken.kraken and JP4D.report.

Let’s look at the precomputed outputs of kraken2 for our JP4D reads.

head ~/dc_workshop/taxonomy/JP4D.kraken

U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:19691:2037	0	250|251	0:216 |:| 0:217
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:14127:2052	0	250|238	0:216 |:| 0:204
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:14766:2063	0	251|251	0:217 |:| 0:217
C	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:15697:2078	2219696	250|120	0:28 350054:5 1224:2 0:1 2:5 0:77 2219696:5 0:93 |:| 379:4 0:82
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:15529:2080	0	250|149	0:216 |:| 0:115
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:14172:2086	0	251|250	0:217 |:| 0:216
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:17552:2088	0	251|249	0:217 |:| 0:215
U	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:14217:2104	0	251|227	0:217 |:| 0:193
C	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:15110:2108	2109625	136|169	0:51 31989:5 2109625:7 0:39 |:| 0:5 74033:2 31989:5 1077935:1 31989:7 0:7 60890:2 0:105 2109625:1
C	MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:19558:2111	119045	251|133	0:18 1224:9 2:5 119045:4 0:181 |:| 0:99


This information may be confusing, let’s take out our cheatsheet to understand some of its components:

Column Description
C Classified or unclassified
MISEQ-LAB244-W7:156:000000000-A80CV:1:1101:15697:2078 FASTA header of the sequence
2219696 Tax ID
0:28 350054:5 1224:2 0:1 2:5 0:77 2219696:5 0:93 379:4 0:82 kmers hit to a taxonomic ID e.g. tax ID 350054 has 5 hits, tax ID 1224 has 2 hits, etc.

As we can see, the kraken file is not very readable. So let’s look at the report file:

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
head ~/dc_workshop/taxonomy/JP4D.report

 78.13	587119	587119	U	0	unclassified
21.87	164308	1166	R	1	root
21.64	162584	0	R1	131567	  cellular organisms
21.64	162584	3225	D	2	    Bacteria
18.21	136871	3411	P	1224	      Proteobacteria
14.21	106746	3663	C	28211	        Alphaproteobacteria
7.71	57950	21	O	204455	          Rhodobacterales
7.66	57527	6551	F	31989	            Rhodobacteraceae
1.23	9235	420	G	1060	              Rhodobacter
0.76	5733	4446	S	1063	                Rhodobacter sphaeroides


## Taxonomic assignment of the contigs of a MAG

We now have the taxonomic identity of the reads of the whole metagenome, but we do not know to which taxon our MAGs correspond to. For this we have to make the taxonomic assignment with their contigs instead of its reads, because we do not have the reads that correspond to a MAG separated from the reads of the entire sample.

For this the kraken2 is a little bit different; here we can look at the command for the JP4D.001.fasta MAG:

$mkdir TAXONOMY_MAG$ kraken2 --db kraken-db --threads 12 -input JP4D.001.fasta --output TAXONOMY_MAG/JP4D.001.kraken --report TAXONOMY_MAG/JP4D.001.report


The results of this are pre-computed in the ~/dc_workshop/taxonomy/mags_taxonomy/ directory

$cd ~/dc_workshop/taxonomy/mags_taxonomy$ ls

JP4D.001.kraken
JP4D.001.report

more ~/dc_workshop/taxonomy/mags_taxonomy/JP4D.001.report

 50.96	955	955	U	0	unclassified
49.04	919	1	R	1	root
48.83	915	0	R1	131567	  cellular organisms
48.83	915	16	D	2	    Bacteria
44.40	832	52	P	1224	      Proteobacteria
19.37	363	16	C	28216	        Betaproteobacteria
16.22	304	17	O	80840	          Burkholderiales
5.66	106	12	F	506	            Alcaligenaceae
2.72	51	3	G	517	              Bordetella
1.12	21	21	S	2163011	                Bordetella sp. HZ20
.
.
.


By looking at the report, we can see that half of the contigs are unclassified, and that a very little proportion of contigs have been assigned an OTU. This is weird because we expected to have only one genome in the bin.

Just to exemplify how a report of a complete and not contaminated MAG should look like, let’s look at the report of this MAG from another study:

100.00	108	0	R	1	root
100.00	108	0	R1	131567	  cellular organisms
100.00	108	0	D	2	    Bacteria
100.00	108	0	P	1224	      Proteobacteria
100.00	108	0	C	28211	        Alphaproteobacteria
100.00	108	0	O	356	          Rhizobiales
100.00	108	0	F	41294	            Bradyrhizobiaceae
100.00	108	0	G	374	              Bradyrhizobium
100.00	108	108	S	2057741	                Bradyrhizobium sp. SK17


## Discussion:

Why do you think we found so many OTUs in this bin?

## Visualization of taxonomic assignment results

After we have the taxonomy assignation what follows is some visualization of our results. Krona is a hierarchical data visualization software. Krona allows data to be explored with zooming, multi-layered pie charts and includes support for several bioinformatics tools and raw data formats. To use Krona in our results, let’s go first into our taxonomy directory, which contains the pre-calculated Kraken outputs.

### Krona

With Krona we will explore the taxonomy of the JP4D.001 MAG.

$cd ~/dc_workshop/taxonomy/mags_taxonomy  Krona is called with the ktImportTaxonomy command that needs an input and an output file. In our case we will create the input file with the columns three and four from JP4D.001.kraken file. $ cut -f2,3 JP4D.001.kraken > JP4D.001.krona.input


Now we call Krona in our JP4D.001.krona.input file and save results in JP4D.001.krona.out.html.

$ktImportTaxonomy JP4D.001.krona.input -o JP4D.001.krona.out.html  Loading taxonomy... Importing JP4D.001.krona.input... [ WARNING ] The following taxonomy IDs were not found in the local database and were set to root (if they were recently added to NCBI, use updateTaxonomy.sh to update the local database): 1804984 2109625 2259134  And finally, open another terminal in your local computer,download the Krona output and open it on a browser. $ scp dcuser@ec2-3-235-238-92.compute-1.amazonaws.com:~/dc_workshop/taxonomy/JP4D.001.krona.out.html .


You will see a page like this:

## Exercise 2: Exploring Krona visualization

Try double clicking on the segment of the pie chart that represents Bacteria and see what happens. What percentage of bacteria is represented by the genus Paracoccus?

Hint: There is a search box in the top left corner of the window.

## Solution

2% of Bacteria corresponds to the genus Paracoccus in this sample. In the top right of the window we see little pie charts that change whenever we change the visualization to expand certain taxa.

### Pavian

Pavian is another visualization tool that allows comparison between multiple samples. Pavian should be locally installed and needs R and Shiny, but we can try the Pavian demo WebSite to visualize our results.

First we need to download the files needed as inputs in Pavian, t his time we will visualize the assignation of the reads of both samples: JC1A.report and JP4D.report.
This files corresponds to our Kraken reports. Again in our local machine lets use scp command.

\$ scp dcuser@ec2-3-235-238-92.compute-1.amazonaws.com:~/dc_workshop/taxonomy/*report .


We go to the Pavian demo WebSite, click on Browse and choose our reports. You need to select both reports at the same time.

We click on the Results Overview tab.

We click on the Sample tab.

We can look at the abundance of a specific taxon by clicking on it.

We can look at a comparison of both our samples in the Comparison tab.

## Discussion: Taxonomic level of assignment

What do you think is harder to assign, a species (like E. coli) or a phylum (like Proteobacteria)?

## Key Points

• A database with previous gathered knowledge (genomes) is needed for taxonomic assignment.

• Taxonomic assignment can be done using Kraken.

• Krona and Pavian are web based tools to visualize the assigned taxa.