11  Authentication of the pathogens

11.1 Introduction

In this part, we will start authenticating the pathogens found by the krakenuniq tool. This part is complex and needs database dependencies to work. So we will focus only one microbe (taxid 2047) from the sample sample1.

We will run an sbatch script, and we will check the outputs. Before working on this part, lets export the PATH variable to check the output files:

export PATH=${PATH}:/truba/home/egitim/miniconda3/envs/aMeta/bin/

The main logic in this section, is to:

  • Get information of the scientific name and sequence id information of the particular microbe
  • Extract DNA reads assigned to one specific microbe
  • Calculate several authentication parameters from the sample1.trimmed.rma6 file
  • Create a bam file from the sample1.trimmed.sam.gz using the sequence id of the microbe
  • Extract read length, breadth of coverage, and post-mortem decay parameters from the sam file
  • Combine these information into nice looking pdf file
  • Calculate the authenticatio score for the particular microbe

First, let’s run the sbatch script, and then we will start checking the main output files while it is running:

sbatch Authentic.sh --account=egitim

## Scientific name and sequence ID extraction

Let’s go step by step.

In the krakenuniq part, we created a file called taxID.pathogens.

Let’s check this file:

less /truba/home/egitim/aMeta/results/KRAKENUNIQ/sample1/taxID.pathogens

Afterwards, we will extract the node name from the krakenuniq database. We can not show the output, because it needs the big krakenuniq database.

Let’s check the output:


less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/node_list.txt

THis pathogen name is Rothia dentocariosa. Over the next steps, we will extract DNA reads assigned to this pathogen, and we will create authenticity metrics.

Then we will extract the sequence name of the reference sequence of the bacteria from the database:

/truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/name_list.txt

11.2 DNA read extraction and postprocessing

Then we will use MaltExtract and postprocessing.AMPS.r tools to extract DNA reads assigned to this pathogen, from the rma6 file of the sample1.

Let’s check this folder:


ls /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/MaltExtract_output/

The ancient folder contains statistics only for ancient DNA reads, and default folder contains statistics for all DNA reads.

If we check the default folder, we can see that several parameters are organized into sub folders:

s /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/MaltExtract_output/modern/

11.3 Creating a sam file for the microbe of interest

In this step, we extract alignment entries from the malt sam file using this sequence ID that we previously extracted,

Let’s check the output file:

samtools view /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/sorted.bam | less

From this file, we will extract breadth of coverage and read length distribution information:

less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/breadth_of_coverage
less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/read_length.txt

Then we extract DNA sequence of the reference file to use with IGV tool:

less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/CP009643.1.fasta

We calculate PMD scores:

less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/PMDscores.txt

11.4 Combine authentication parameters and score

Using the authentic.R script, we create the last authentication plot:

ls /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/authentic_Sample_sample1.trimmed rma6_TaxID_2047.pdf

Lets check the authentication plot:

Authentication plot for the bacteria

And at last, authentication scores:


less /truba/home/egitim/aMeta/results/AUTHENTICATION/sample1/2047/authentication_scores.txt

The script should have finished by now. Let’s check the output folder:

ls results/AUTHENTICATION/sample1/2047