Samuel J Gurr, PhD

AOX Pgenerosa identification

by Samuel J Gurr PhD 2 min read

OBJECTIVE

The following protocol was completed with the geoduck genome in preparation for primer design and qPCR of a target protein Alternative oxidase This step-by-step process can be used to verify the presence/absence of a particular target gene within an annotated genome without gene family identification. In this case.. a database of the geoduck genome was used with only ID identifiers for genes (no family-specific accession ID)

1.) Create a fasta file query

### Objective: create a query of the nucelotide seq data AOX_seq.fasta

  • targetted bivalve taxa and alternative oxidase - complied ~8 sequences from NCBI into a fasta file NOTE: txt format can be converted into fasta by opening the file in sublime, notepad++ etc. and :savea.” .fasta”

    2.) Make a blast database

    Objective: download the P.generosa reference genome (.fna) and convert to a blast database

    SYNTAX in terminal make blast database (nucleotides)

  • makeblastdb.exe
  • -in ~/Documents/genomes/Panopea-generosa-genes.fna
  • -dbtyp nucl
  • -title Pgenerosa.GENES
  • -out ~/Documents/genomes/Pgenerosa_database/Pgenerosa.GENES
  • -parse_seqids

    SYNTAX in terminal blastn (nucelotide) AOX_seq.fasta to the whole annotated database

  • blastn
  • -db ~/Documents/genomes/Pgenerosa_database/Pgenerosa.GENES
  • -query
  • -out ~/Documents/blastdb/AOX_blastn_GENES.txt
  • -evalue 0.001
  • -word_size 11
  • -dust no
  • -num_alignments 1
  • -num_descriptions 5
  • -out haktan.txt
  • -dust no
  • -task blastn

NOTE: add -outfmt 6 == qsequid ssequid pident length mismatch gapopen qstart qend sstart send evalue bitscore; -outfmt 11 == archive format; -num_descriptions 5 == the five seqeunces with the highest bitscore

3.) View output

Objective: determine the gene(s) representative of the query/protein target (i.e. AOX_seq.fasta)

PGEN_.00g108770 is the best hit!

4.) Use Muscle in Jalview

Objective:

view the regions of overlap between the PGEN_.00g108770 gene and the AOX_seq fasta

Putative exon regions!

  • many regions without overlap – likley untraslated regions (UTRs)

UTRs

4.) Augustus

Objective:

run sequence against a similar or related reference – the reference applies a criteria of untraslated v. translated regions to identify and compile the coding region of an uploaded sequence.

  • upload the PGEN_.00g108770 nucelotide sequence

  • apply Pisaster as the reference (sea star)

NOTE: the output from Augustus give you your full coding sequence (nucleotides) and amino acid seq data for the putative full exon region (i.e. Pisaster as ref criteria) Augustus gff output

4.) Pfam (Augustus follow-up)

Objective:

determine the protein family of the protein sequence output from Augustus.

  • copy the protein sequence (nucleotide will give the same result) and paste into Pfam “Sequence Search” - press Go

  • high evidence that PGEN_.00g108770 is AOX! Focus on cover via “to and “end” of the Hidden-Markov-Model in relation to the HMM “length”

Pfam result

5.) NCBI blastp (Augustus follow-up)

Objective:

determine the the sequences within NCBI with the highest %cover and %identity to your putative exon sequence from Augustus NOTE: remember we used Pisaster as a reference criteria in Augustus to map UTRs and exons!

  • copy the protein sequence and run blastp in NCBI

  • high evidence that PGEN_.00g108770 is AOX and related to the bivalve taxa!!!

NCBI blastp result