Taxonomic classification tools match sequences - typically reads or assembled contigs - against a database of microbial genomes to identify the taxon of each sequence. In the early days of metagenomics, the best strategy was to use BLAST to compare each read with all sequences in GenBank. As the reference databases and the size of sequencing data sets have grown, alignment using BLAST has become computationally infeasible, leading to the development of metagenomics classifiers that provide much faster results, although usually with less sensitivity than BLAST. A variety of strategies have been used for the matching step: aligning reads, mapping k-mers, using complete genomes, aligning marker genes only or translating the DNA and aligning to protein sequences.
OmicsBox comes with Kraken as the tool of choice for taxonomic classification because of its overall positive characteristics, such as being 16S and WGS capable and showing good benchmark scores.
Kraken is a taxonomic sequence classifier that assigns taxonomic labels to short DNA reads. It does so by examining the k-mers within a read and querying a database with those k-mers. This database contains a mapping of every k-mer in Kraken's genomic library to the lowest common ancestor (LCA) in a taxonomic tree of all genomes that contain that k-mer. The set of LCA taxa that correspond to the k-mers in a read are then analyzed to create a single taxonomic label for the read; this label can be any of the nodes in the taxonomic tree. Kraken is designed to be rapid, sensitive, and highly precise. This approach is feasible for metagenomics WGS as well as 16S/ITS amplicon read input data.
The current Kraken database version is 2019.06 and was created by us. The contained species and strains are listed in this file and can be opened through File > Load > Load Kraken Data to explore the database content (Figure 1 and Figure 2).
- Sequencing Data: Choose the type of input data: single-end, paired-end or interleaved paired-end reads If paired-end is selected, two files per sample are required and the file pattern has to be provided.
- Reads, Contigs or Genes: Select files that contain the desired input data. Kraken was designed to work with short reads, but works reliable with assembled sequences or genes.
- Paired-end configuration: When working with paired-end libraries, a so-called pattern has to be established to help the software distinguish between upstream and downstream read files. Per default, we assume the following pattern:
- upstream: SampleA_1.fastq
- downstream: SampleA_2.fastq
For example, if the upstream file is named SRR037717_1.fastq and the downstream one SRR037717_2.fastq, you should establish "_1" as the upstream pattern and "_2" as the downstream pattern.
Figure 1. Taxonomic Classification Wizard: input page.
Metagenome sequence data is often "contaminated" by the host organism, e.g. a human gut genome sequencing project will contain reads from Homo sapiens. Therefore, Kraken can be combined with a prior contaminant screening to filter out reads that may stem from a host organism. We offer various model organism genomes, the screening is performed with the help of Bowtie2.
- Homo sapiens
- Arabidopsis thaliana
- Bos taurus
- Drosophila melanogaster
- Escherichia coli
- Mus musculus
- Rattus norvegicus
- Sus scrofa
Figure 2. Taxonomic Classification Wizard: configuration page.
The results of the taxonomic classification with Kraken are:
- Main result table, that shows all identified OTUs for each provided sample.
- PDF Report that shows overall input and carried-out analysis information.
- Stacked bar chart to compare samples at specific taxonomic levels.
- Radial cladogram in form of a Krona chart to study OTU abundances in a sample.
- Rarefaction curves to assess the sequencing depth.
- Chao1 (species) diversity curve to evaluate the (species) diversity in the whole data-set.
- Principal Coordinates Analysis plot to get an overview and to identify outliers.
Main result table
The result table (Figure 3) shows for each analyzed sample the number of OTUs found and their confidence scores. OTU stands for operational taxonomic unit. A count of 0 means the OTU is not present in the current sample. The evidence scores vary from 0 to 1, where 1 is best. The counts shown are cumulative, meaning that they do not only show direct hits for a certain OTU, but also sum up OTUs that converge into them in the taxonomic tree i.e. the taxonomic tree's hierarchy is taken into account. We use the taxonomic hierarchy from the NCBI in a simplified form and it consists of 8 main levels instead of 33, i.e. the many levels are summarized as shown in Figure 4.
The result table can be used to filter for certain taxonomic levels via the column header filter function, e.g. showing only the species or phylum level OTUs.
Right clicking an OTU shows the table context menu to create statistics and id lists, and provides an extra functionality. The Extract Sequences option allows to export the read names and actual reads of the currently selected OTUs. This makes it possible to export all reads that have been classified as e.g. bacteria and thus reduce the dataset size for further gene finding and functional annotation. Many other scenarios and use cases exist.
Figure 3. Taxonomic Classification results table.
Figure 4. NCBI taxonomic hierarchy.
Stacked Bar Chart
The Stacked bar chart (Figure 5) is a combined view for inter sample comparison, separated in the 7 main taxonomic levels. Average OTUs are ordered by abundance from high to low. Only the 500 biggest OTUs are shown for each sample, the remaining are gathered into an extra group called Others. These low frequent OTUs can be analyzed in detail with the Krona Pie Chart. The button Hide Unclassified in the top-right corner shows how the percentages change when only taking into account the data that could be classified by Kraken.
The graphic can be exported as a PNG image by clicking the corresponding icon in the top-right corner.
Figure 5. Stacked bar chart.
Krona Pie Chart
This graphic (Figure 6) shows a slightly modified Krona chart with various options in the sidepanel. Again, the counts are cumulative and grouped into roughly 8 main levels. However, all direct counts are shown as well, which is helpful when looking at the "below species" level, which includes subspecies and strains.
The currently visualized sample is selected from a list in the sidepanel, the All Combined entry shows all samples together in one chart. Furthermore, text sizes can be adjusted and OTUs can be searched. Coloring by average Kraken evidence scores is also possible.
The graphic can be exported as a PNG image and PDF by clicking the corresponding icons in the top-right corner.
A summary report which shows basic statistics and alpha-diversity indices for each analyzed sample. It also gives information about the percentages of reads that were classified. In addition, for each of the 7 main taxonomic levels (Superkingdom, Phylum, Class, Order, Family, Genus and Species), the top 10 OTUs per sample are listed.
The graphic can be exported as PDF by clicking the corresponding icon in the top-right corner.
Figure 6. Krona pie chart.
In addition, more charts and statistics can be generated to offer a global visualization of the taxonomic classification results. These charts can be found in the sidepanel of the taxonomic classification results.
Rarefaction is a technique widely used in ecology which is applied to OTU analysis. A rarefaction graph (Figure 7) represents the number of expected OTUs (Y-axis) found in n NGS reads (X-axis).
The goal of rarefaction is to determine whether sequencing coverage is deep enough to get a good estimate of the total number of the OTUs present in a specific sample.
If the rarefaction curve still presents a growing trend at its end, it means that the coverage is not enough to adequately represent the real microbial diversity of the sample. By contrast, if the curve shows a horizontal asymptote, it means that a good estimation of diversity was obtained.
Note that the results of the rarefaction technique give us a suggestion about the coverage, but they are not conclusive, i.e. rare OTUs that are present in the sample could not be yet observed even if the curve presents an asymptotic trend.
Figure 7. Rarefaction curves.
An accumulation or diversity curve (Figure 8) plots the cumulative number of distinct OTUs discovered as a function of the number of samples examined. I.e. The minimum, average and maximum number of OTUs, when looking at 1, 2, ... N samples of the current dataset.
This curve can be used to evaluate the benefits in microbial diversity of including additional samples to the dataset, or to compare this diversity across datasets with similar sampling efforts.
Figure 8. Diversity curve.
Principal Coordinate Analysis (PCoA Plot)
PCoA (Figure 9) is a two-dimensional plot in which the Bray-Curtis distances between samples are drawn. You can select an experimental condition to color the points from an experimental design, and the taxonomy level from which the distances will be calculated.
Figure 9. PCoA plot.