July / August, 2024

Developed by:
Egon A. Ozer, MD PhD ([email protected])
Ramon Lorenzo Redondo, PhD ([email protected])

Using long-read nanopore sequenncing we will assemble a SARS-CoV-2 sample, generate a variant report, and a consensus. We will then check our consensus and the one generated with Illumina to asign the clade and lineage of the virus.

We will practice a command line assembly and analysis. We will use minimap2 (https://github.com/lh3/minimap2) that is one of the best options for long-read assemblies. Minimap2 will generate a sam file that we will process similar to yesterday's with samtools to generate a sorted and indexed bam file.

We activate the environment:

micromamba deactivate

micromamba activate nanopore

And run the pipeline

cd ~/sl_workshop_2024/demo_data/nanopore_reads

minimap2 \
  -ax map-ont \
  ~/sl_workshop_2024/demo_data/reference/nCoV-2019.reference.fasta \
  COV07735.nanopore.fastq.gz > COV07735.nanopore.sam

samtools view -bS -F 4 COV07735.nanopore.sam > COV07735.nanopore.bam

samtools sort -o COV07735.nanopore_sorted.bam COV07735.nanopore.bam

samtools index COV07735.nanopore_sorted.bam

Activate the alignment environment

micromamba deactivate

micromamba activate alignment

Trim primers

ivar trim \
	-q 0 \
	-i COV07735.nanopore_sorted.bam \
	-b ~/sl_workshop_2024/demo_data/reference/nCoV-2019.ivar.bed \
	-p COV07735.trimmed
samtools sort -o COV07735.trimmed_sorted.bam COV07735.trimmed.bam

samtools index COV07735.trimmed_sorted.bam

Generate pileup file

samtools mpileup \
	-aa \
	-A \
	-d 0 \
	-Q 0 \
	--reference ~/sl_workshop_2024/Downloads/demo_data/reference/nCoV-2019.reference.fasta \
	COV07735.trimmed_sorted.bam \
	> COV07735.pileup.txt

Call variants based on pileup output

ivar variants \
	-p COV07735.variants \
	-t 0.03 \
	< COV07735.pileup.txt

ivar consensus \
	-m 10 \
	-q 0 \
	-t 0 \
	-p COV07735.consensus \
	< COV07735.pileup.txt

We will now use Nextclade to assign the clade and the quality of our consensus.

1. Go to https://clades.nextstrain.org/

   

2. Select the SARS-CoV-2 reference

   

3. Load our COV07735.consensus.fa and analyze

   

Let's discuss the results check the tree and repeat with our the SAR_CoV_2 consensus sequence from Illumina reads we generated earlier.

We will check Nextrain web to see the possible analyses we can perform that will be covered tomorrow in Phylogenetics

• Go to https://nextstrain.org/

This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.