Samenvatting

Summary Bioinformatics 1e Master - Evolution

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Katholieke Universiteit Leuven (KU Leuven)

Alle commando's van de 5 lessen met uitleg, voorbeeldoefeningen uitgewerkt, proef examen uitgewerkt

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Bio informatics: Evolution

Session 1: Database querying with BLAST, multiple sequence alignment
and phylogenetic reconstruction
Jupyter notebook: exerciseYFV.blast.search.AliView
- AliView: not on the exam
Goal of this session:
- Query GenBank database via web interface and through command line
- Look up sequences similar to a query in a database with BLAST via the
command line
- create and edit a multiple sequence alignment
- reconstruct a phylogeny with the NJ algorithm

Workflow:
- Look up patients virus sequence => YFV sequence
- Fetch a set of highly similar sequences
- Create and edit multiple sequence alignment
- Reconstruct NJ tree
The goal is to investigate the genetic relationship of a patient’s genome with publicly
available sequences (focusing on samples from 1 outbreak), to determine whether
the patient’s virus strain is more closely related to strains from outbreak or if it
represents a district lineage from a different region.
1) Load Python modules to be used
= from Bio.Blast import NCBIWWW, NCBIXML
from Bio import Entrez, SeqIO, Phylo, AlignIO
import pandas as pd
import time, os, alv, re
from IPython.display import Image
*Change nothing*
2) Load interface to run R embedded in a Python process:
= %load_ext rpy2.ipython
3) Load R packages to be used:
= %%R

,library("ape")
4) Define variables for input names that will only will have to be changed once
= baseName = "namefile(without .fasta)"
 Base name is main part of the filename without details
= folderName = "exercise_xxx"
 Name of directory where these files are stored

5) Push that variable to R using Rpush
= %Rpush baseName folderName
6) Check if this works:
= %%R
print(paste0("baseName: ", baseName, " -- folderName: ", folderName))
7) Let’s check this for Bash:
= %%bash -s "$baseName" "$folderName"
echo baseName: "$1" -- folderName: "$2"
8) Create a folder where analysis files can be saved:
= %%bash -s "$folderName"
= mkdir "$1"
 If folder already exists, try $mkdir -p "$1
Database querying
First step = look up patient’s sequence in GenBank with accession number MF347613
=> download it => browse to https://www.ncbi.nlm.nih.gov/nuccore and save file as
fasta-format => upload in Jupyter
(1) Go to GenBank: paste accession number in search bar

(2) Download as fasta & change name
(3) Upload in Jupyter

, 1) Move uploaded file into the folder for this exercise or upload sequence file to the
exercise folder (skip this command then)
= %%bash -s "$baseName" "$folderName"
mv ${1}.fasta ${2}/${1}.fasta

2) Change directory to exercise’s folder
= os.chdir(folderName)
3) Use query sequence to fetch the 100 most similar sequence available in NCBI
nucleotide database:
= inputFile = baseName+'.fasta' #your sequence file
query = SeqIO.read(inputFile, format="fasta") #Read sequence
search_type = "blastn"
database = "nt"
max_nr_hits = 100 #Adjust if you want more/less results
before = round(time.time(), ndigits=0)
blast_stream = NCBIWWW.qblast(program = search_type,
database = database,
sequence = query.seq,
alignments = 1,
hitlist_size = max_nr_hits)
after = round(time.time(), ndigits=0)
text = f"The BLAST search took {after - before} seconds."
print(text)
 This script performs BLAST search using NCBIWWW.qblast function from
Biopython, which queries the NCBI BLAST database
 Defines input file
 Reads query sequence: uses SeqIO.read() from Biopython to read a FASTA file
& stores the sequence object in query
 Uses BLASTN for nucleotide sequence comparison
 Searches against NCBI’s nucleotide database
 Limits results to 100 matches
 Stores current time before BLAST search starts
 NCBIWWW.qblast() submits the query sequence (query.seq) to NCBI’s online
BLAST service

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