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Kraken2

Quick Facts

Workflow Type Applicable Kingdom Last Known Changes Command-line Compatibility Workflow Level
Standalone Any Taxa PHB v2.3.0 Yes Sample-level

Kraken2 Workflows

The Kraken2 workflows assess the taxonomic profile of raw sequencing data (FASTQ files).

Kraken2 is a bioinformatics tool originally designed for metagenomic applications. It has additionally proven valuable for validating taxonomic assignments and checking contamination of single-species (e.g. bacterial isolate, eukaryotic isolate, viral isolate, etc.) whole genome sequence data.

There are three Kraken2 workflows:

  • Kraken2_PE is compatible with Illumina paired-end data
  • Kraken2_SE is compatible with Illumina single-end data
  • Kraken2_ONT is compatible with Oxford Nanopore data

Besides the data input types, there are minimal differences between these two workflows.

Kraken2 Workflow Diagram

Kraken2 Workflow Diagram

Databases

Database selection

The Kraken2 software is database-dependent and taxonomic assignments are highly sensitive to the database used. An appropriate database should contain the expected organism(s) (e.g. Escherichia coli) and other taxa that may be present in the reads (e.g. Citrobacter freundii, a common contaminant).

Suggested databases

Database name Database Description Suggested Applications GCP URI (for usage in Terra) Source Database Size (GB) Date of Last Update
Kalamari v5.1 Kalamari is a database of complete public assemblies, that has been fine-tuned for enteric pathogens and is backed by trusted institutions. Full list available here ( in chromosomes.tsv and plasmids.tsv) Single-isolate enteric bacterial pathogen analysis (Salmonella, Escherichia, Shigella, Listeria, Campylobacter, Vibrio, Yersinia) gs://theiagen-large-public-files-rp/terra/databases/kraken2/kraken2.kalamari_5.1.tar.gz ‣ 1.5 18/5/2022
standard 8GB Standard RefSeq database (archaea, bacteria, viral, plasmid, human, UniVec_Core) capped at 8GB Prokaryotic or viral organisms, but for enteric pathogens, we recommend Kalamari gs://theiagen-large-public-files-rp/terra/databases/kraken2/k2_standard_08gb_20240112.tar.gz https://benlangmead.github.io/aws-indexes/k2 7.5 12/1/2024
standard 16GB Standard RefSeq database (archaea, bacteria, viral, plasmid, human, UniVec_Core) capped at 16GB Prokaryotic or viral organisms, but for enteric pathogens, we recommend Kalamari gs://theiagen-large-public-files-rp/terra/databases/kraken2/k2_standard_16gb_20240112.tar.gz https://benlangmead.github.io/aws-indexes/k2 15 12/1/2024
standard Standard RefSeq database (archaea, bacteria, viral, plasmid, human, UniVec_Core) Prokaryotic or viral organisms, but for enteric pathogens, we recommend Kalamari gs://theiagen-large-public-files-rp/terra/databases/kraken2/k2_standard_20240112.tar.gz https://benlangmead.github.io/aws-indexes/k2 72 18/4/2023
viral RefSeq viral Viral metagenomics gs://theiagen-large-public-files-rp/terra/databases/kraken2/k2_viral_20240112.tar.gz https://benlangmead.github.io/aws-indexes/k2 0.6 12/1/2024
EuPathDB48 Eukaryotic pathogen genomes with contaminants removed. Full list available here Eukaryotic organisms (Candida spp., Aspergillus spp., etc) gs://theiagen-public-files-rp/terra/theiaprok-files/k2_eupathdb48_20201113.tar.gz https://benlangmead.github.io/aws-indexes/k2 30.3 13/11/2020
EuPathDB48 Eukaryotic pathogen genomes with contaminants removed. Full list available here Eukaryotic organisms (Candida spp., Aspergillus spp., etc) gs://theiagen-large-public-files-rp/terra/databases/kraken/k2_eupathdb48_20230407.tar.gz https://benlangmead.github.io/aws-indexes/k2 11 7/4/2023

Inputs

Terra Task Name Variable Type Description Default Value Terra Status Workflow
*workflow_name kraken2_db File A Kraken2 database in .tar.gz format Required ONT, PE, SE
*workflow_name read1 File Required ONT, PE, SE
*workflow_name read2 File Required for PE only PE
*workflow_name samplename String Required ONT, PE, SE
kraken2_pe or kraken2_se classified_out String Allows user to rename the classified FASTQ files output. Must include .fastq as the suffix classified#.fastq Optional ONT, PE, SE
kraken2_pe or kraken2_se cpu Int Number of CPUs to allocate to the task 4 Optional ONT, PE, SE
kraken2_pe or kraken2_se disk_size Int GB of storage to request for VM used to run the kraken2 task. Increase this when using large (>30GB kraken2 databases such as the "k2_standard" database) 100 Optional ONT, PE, SE
kraken2_pe or kraken2_se docker String The Docker container to use for the task us-docker.pkg.dev/general-theiagen/staphb/kraken2:2.1.2-no-db Optional ONT, PE, SE
kraken2_pe or kraken2_se kraken2_args String Allows a user to supply additional kraken2 command-line arguments Optional ONT, PE, SE
kraken2_pe or kraken2_se memory Int Amount of memory/RAM (in GB) to allocate to the task 32 Optional ONT, PE, SE
kraken2_pe or kraken2_se unclassified_out String Allows user to rename unclassified FASTQ files output. Must include .fastq as the suffix unclassified#.fastq Optional ONT, PE, SE
krona cpu Int Number of CPUs to allocate to the task 4 Optional PE, SE
krona disk_size Int Amount of storage (in GB) to allocate to the task 100 Optional PE, SE
krona docker String The Docker container to use for the task us-docker.pkg.dev/general-theiagen/biocontainers/krona:2.7.1--pl526_5 Optional PE, SE
krona memory Int Amount of memory/RAM (in GB) to allocate to the task 8 Optional PE, SE
kraken2_recalculate_abundances cpu Int Number of CPUs to allocate to the task 4 Optional ONT
kraken2_recalculate_abundances disk_size Int Amount of storage (in GB) to allocate to the task 100 Optional ONT
kraken2_recalculate_abundances docker String The Docker container to use for the task us-docker.pkg.dev/general-theiagen/theiagen/terra-tools:2023-08-28-v4 Optional ONT
kraken2_recalculate_abundances memory Int Amount of memory/RAM (in GB) to allocate to the task 8 Optional ONT
kraken2_recalculate_abundances target_organism String Target organism for the kraken2 abundance to be exported to the data table Optional ONT
version_capture docker String The Docker container to use for the task "us-docker.pkg.dev/general-theiagen/theiagen/alpine-plus-bash:3.20.0" Optional ONT, PE, SE
version_capture timezone String Set the time zone to get an accurate date of analysis (uses UTC by default) Optional ONT, PE, SE

Outputs

Variable Type Description
kraken2_classified_read1 File FASTQ file of classified forward/R1 reads
kraken2_classified_read2 File FASTQ file of classified reverse/R2 reads (if PE)
kraken2_classified_report File Standard Kraken2 output report. TXT filetype, but can be opened in Excel as a TSV file
kraken2_docker String Docker image used to run kraken2
kraken2_*_wf_analysis_date String Date the workflow was run
kraken2_*_wf_version String Workflow version
kraken2_report File TXT document describing taxonomic prediction of every FASTQ record. This file is usually very large and cumbersome to open and view
kraken2_unclassified_read1 File FASTQ file of unclassified forward/R1 reads
kraken2_unclassified_read2 File FASTQ file of unclassified reverse/R2 reads (if PE)
kraken2_version String kraken2 version
krona_docker String Docker image used to run krona (if PE or SE)
krona_html File HTML report of krona with visualisation of taxonomic classification of reads (if PE or SE)
krona_version String krona version (if PE or SE)

Interpretation of results

The most important outputs of the Kraken2 workflows are the kraken2_report files. These will include a breakdown of the number of sequences assigned to a particular taxon, and the percentage of reads assigned. A complete description of the report format can be found here.

When assessing the taxonomic identity of a single isolate's sequence, it is normal that a few reads are assigned to very closely rated taxa due to the shared sequence identity between them. "Very closely related taxa" may be genetically similar species in the same genus, or taxa with which the dominant species have undergone horizontal gene transfer. Unrelated taxa or a high abundance of these closely related taxa is indicative of contamination or sequencing of non-target taxa. Interpretation of the results is dependent on the biological context.

Example Kraken2 report

Below is an example kraken2_report for a Klebsiella pneumoniae sample. Only the first 30 lines are included here since rows near the bottom are often spurious results with only a few reads assigned to a non-target organism.

From this report, we can see that 84.35 % of the reads were assigned at the species level (S in the 4th column) to "Klebsiella pneumoniae". Given almost 6 % of reads were "unclassified" and ~2 % of reads were assigned to very closely related taxa (in the Klebsiella genus), this suggests the reads are from Klebsiella pneumoniae with very little -if any- read contamination. 

 5.98    108155 108155  U   0   unclassified
 94.02  1699669 0   C   1   
 94.02  1699669 1862    C1  131567    cellular organisms
 93.91  1697788 2590    D   2       Bacteria
 93.75  1694805 6312    P   1224          Proteobacteria
 93.39  1688284 37464   C   1236            Gammaproteobacteria
 91.31  1650648 35278   O   91347             Enterobacterales
 89.31  1614639 43698   F   543             Enterobacteriaceae
 86.40  1561902 22513   G   570               Klebsiella
 **84.35    1524918 1524918 S   573                 Klebsiella pneumoniae**
  0.75  13596   13596   S   548                 Klebsiella aerogenes
  0.03  600 600 S   244366                  Klebsiella variicola
  0.01  253 253 S   571                 Klebsiella oxytoca
  0.00  17  17  S   1134687                 Klebsiella michiganensis
  0.00  3   0   G1  2608929                 unclassified Klebsiella
  0.00  3   3   S   1972757                   Klebsiella sp. PO552
  0.00  2   2   S   1463165                 Klebsiella quasipneumoniae
  0.17  3035    129 G   590               Salmonella
  0.15  2728    909 S   28901                   Salmonella enterica
  0.03  582 582 S1  9000010                   Salmonella enterica subsp. IIa
  0.02  306 306 S1  59201                     Salmonella enterica subsp. enterica
  0.01  230 230 S1  9000014                   Salmonella enterica subsp. IIIa
  0.01  221 221 S1  9000015                   Salmonella enterica subsp. IIIb
  0.01  136 136 S1  9000016                   Salmonella enterica subsp. IX
  0.01  132 132 S1  9000011                   Salmonella enterica subsp. IIb
  0.01  122 122 S1  59208                     Salmonella enterica subsp. VII
  0.00  41  41  S1  59207                     Salmonella enterica subsp. indica
  0.00  25  25  S1  9000017                   Salmonella enterica subsp. X
  0.00  24  24  S1  9000009                   Salmonella enterica subsp. VIII
  0.01  178 178 S   54736                   Salmonella bongori

Krona visualisation of Kraken2 report

Krona produces an interactive report that allows hierarchical data, such as the one from Kraken2, to be explored with zooming, multi-layered pie charts. These pie charts are intuitive and highly responsive.

Krona will only output hierarchical results for bacterial organisms in its current implementation.

Example Krona report

Below is an example of the krona_html for a metagenomic sample. Taxonomic rank is organised from the centre of the pie chart to the edge, with each slice representing the relative abundance of a given taxa in the sample.

Example Krona Report

Kraken2 Technical Details

Links
Software Source Code Kraken2 on GitHub
Software Documentation https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown
Original Publication(s) Improved metagenomic analysis with Kraken 2