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codemeleon edited this page Aug 14, 2023 · 4 revisions

SeqPanther

Before you start, please README.

Data you have

BAM file(s)

Short reads mapped to a reference genome.

FASTA file

Must contain the reference sequence a user wants to analyze.

GFF files

Must contain the reference sequence a user wants to analyze and have coding regions annotated.

Checking reference sequence id

In a bam file

samtools view -H bam_file.bam | grep ^@SQ | grep 'seq_id'

In a fasta file

grep ">seq_id" fasta_file.fasta

In a gff file

grep "seq_id" gff_file.gff

Note: The sequence id must be the same in all files and must be exact as input provided by a user.

Use Case

Sars-CoV-2

Extracting coordinates with changes

Structuring changes for integration in a reference genome sequence

Manual selection of changes

Integration in a reference genome sequence

HIV Drug Resistance

Files

Bam files

GFF annotation file

FASTA file

Checking genome name in bam files

  • samtools view -H bam_file.bam | grep ^@SQ | grep 'K03455|HIVHXB2CG'
  • Output: @SQ SN:K03455|HIVHXB2CG LN:9719

Checking genome name in gff file

  • grep "K03455|HIVHXB2CG" gff_file.gff
  • Output: K03455|HIVHXB2CG 1 9719
  • Multiple lines are possible
  • Must check that it contains CDS annotation and has region a user is interested in

Checking genome name in fasta file

  • grep "K03455|HIVHXB2CG" fasta_file.fasta
  • Output: >K03455|HIVHXB2CG xxyy dtdt
  • Only one line is expected as output
  • After space information vary based on annotation in fasta

Extracting coordinates with changes

  • Basic command: seqpanther codoncounter -bam data/hiv/bam/Ko48924_K03455_HIVHXB2CG.bam -rid K03455\|HIVHXB2CG -ref data/hiv/K03455.fasta -gff data/hiv/K03455.gff
  • The command will generate 3 files in the current directory: codon_output.csv, indel_output.csv and sub_ouput.csv
  • For more options, run seqpanther codoncounter -h or visit SeqPather Repo
  • Details on the outputs are also available in the README at SeqPather Repo

Structuring changes for integration in a reference genome sequence

  • Command: seqpanther cc2ns -s sub_output.csv -i indel_output.csv
  • The command will generate one file per bam (tsv format). File name will start with bam prefix (before .bam section).

Manual selection of changes

CC2NS

  • Coordinate 2258 and 2375 have multiple type of change. Not required change should be removed. In case of multiple changes, last alteration will be considered.

Integration of changes in a reference genome sequence

Generating consensus sequence

  • Command : samtools index data/hiv/bam/Ko48924_K03455_HIVHXB2CG.bam
  • Command : samtools mpileup -uf data/hiv/K03455.fasta data/hiv/bam/Ko48924_K03455_HIVHXB2CG.bam | bcftools call -c | vcfutils.pl vcf2fq > Ko48924_K03455_HIVHXB2CG.fastq
  • Command : seqtk seq seq -aQ64 Ko48924_K03455_HIVHXB2CG.fastq > consensus/Ko48924_K03455_HIVHXB2CG.fasta

Integrating changes

  • Command: seqpanther nucsubs -r data/hiv/K03455.fasta -c consensus -t changes -o output -i K03455\|HIVHXB2CG
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