Error Correction using Medaka

Error correction and polishing are methods to reduce the number of errors in your assembly that come from the relatively noise long-read Oxford Nanopore reads. Althouh Oxford Nanopore reads have constantly improved in sequencing quality they still contain more sequencing/basecalling errors than for example Illumina short reads.

Different algorithms exist for error reduction, including signal-based tools like NanoPolish, and read-based tools like Racon and Medaka. Often several polishing tools are combined, e.g., 3 rounds of Racon and one round of Medaka.

Here, we will use Medaka on the Miniasm and Flye assemblies and see how they perform.

Error correction of the miniasm assembly

Change in the miniasm-minimap directory int he assembler_practical directory and call the tool medaka_consensus on the miniasm.fasta. The read sequences in the filtered.fastq file will be used by medaka to infer sequencing errors to be corrected. In addition to the reads and the assembly medaka requires you to chose the nanopore chemistry and basecalling model the reads were produced on. In our case the reads were produced on a R10.4 flowcell and basecalled with a super-accuracy 400bps model. The output should be written to directory miniasm_medaka_output.

course_user> medaka_consensus -d ./miniasm.fasta \
course_user> -i ../../qc_practical/filtered.fastq \
course_user> -m r1041_e82_400bps_sup_g615 -o miniasm_medaka_output

As with some other steps Medaka is very computationally expensive and will run for a long time. Please stop Medaka pressing Ctrl-c and copy the contents of directory ~/biosec_course/misc/assembler_prac/miniasm_medaka_output/ into the miniasm-medaka-output directory in this directory:

course_user> cp ~/biosec_course/misc/assembler_prac/miniasm_medaka_output/* \
course_user> ./miniasm_medaka_output/

In medaka’s output directory is the polished sequence called consensus.fasta. Compare this file to the B. fermentans reference using dnadiff:

course_user> dnadiff -p miniasm_medaka ~/biosec_course/misc/b_fermentans.fna  \
course_user> ./miniasm_medaka_output/consensus.fasta

Open the report file and have a look at the output

Answer

Error correction of the Flye assembly

Now repeat the same process with the Flye assembly. First change into the assembler_practical/flye directory and call medaka on the assembly.fasta file in the flye_output directory:

course_user> medaka_consensus -d ./flye_output/assembly.fasta -i \
course_user> ../../qc_practical/filtered.fastq -m r1041_e82_400bps_sup_g615 
course_user> -o flye_medaka_output

As before, in case oyu don’t want to wait, stop medaka and copy the contents of ~/biosec_course/misc/assembler_prac/flye_medaka_output into the flye_medaka_output directory here

course_user> cp ~/biosec_course/misc/assembler_prac/flye_medaka_output/* \
course_user> ./flye_medaka_output/

… and run dnadiff on it

course_user> dnadiff -p flye_medaka ~/biosec_course/misc/b_fermentans.fna \
course_user> ./flye_medaka_output/consensus.fasta

Answer

CONTINUE ->