TITLE: Bacterial genome annotation script using BLASTN
AUTHORS: Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

[DESCRIPTION]
This protocol uses a python based script and command-line blastn to annotate Sanger sequencing results from genome amplifications. Its main use in our lab (https://biocomputationlab.com) is to identify the location and gene locus of transposon inserts in microbial bacterial genomes of Pseudomonas putida KT2440. However, this script can be used for other bacterial genomes for which its genome sequence and annotation are available.

Script was developed in python 3.9 with blastn version 2.2.18.

[BEFORE_START]
To run this script command-line blastn and python 3 with packages sys, pandas and os must be installed. 


[GUIDELINES]
This script needs 4 arguments in the following order:
Directory of folder containing sequencing reads in .txt or .seq format
Reads file type (txt or seq)
Genome file to perform blastn alignment (FASTA format)
Genome annotation file (.csv)


[STEPS]
SECTION: Annotation of sequencing reads
2. Run the following python based script with the required arguments


 

 


SECTION: Annotation of sequencing reads
1. Download genome file in FASTA format and annotation file in .csv for the microbial organism to use as reference


 

SECTION: Annotation of sequencing reads
3. Output is a folder named results_script_blast which contains three files:
all_seq_aligned.sam
all_seq_aligned.txt
table_reads_genes_description.csv

SECTION: Example: Annotation of sequencing results from P. putida KT2440
4. Input files

Directory of sequencing reads (it is a zip but shoul be a directory)   In this case the type of file (extension) is txt
Genome in FASTA format   
Annotation file of that genome   
SECTION: Example: Annotation of sequencing results from P. putida KT2440
5. Command-line

  
  

SECTION: Example: Annotation of sequencing results from P. putida KT2440
6. Output files

A new folder named results_script_blast (output files attached in the following zip file) contains a table with information about the alignment and genomic context of each sequencing read.

  

 
                  query acc.s. start% identityalignment lengthmismatchesgap opensevaluebit scoresubject strandLocus TagFeature TypeStartEndStrandGene NameProduct NameSubcellular Localization [Confidence Class]Multiple AllignmentsRest of Locus Tag AssociatedH220707-054_B23_219DZAA034_premix.ab1617023999.04299999999999209111.8599999999999997e-103374plusPP_5408CDS6169113061702940--hypothetical proteinCytoplasmic [Class 3]FALSO-H220707-054_P21_219DZAA035_premix.ab1617023098.618217122.9799999999999995e-106383plusPP_5408CDS6169113061702940--hypothetical proteinCytoplasmic [Class 3]FALSO-H220707-054_L21_219DZAA036_premix.ab1617023099.539217011.4899999999999998e-109394plusPP_5408CDS6169113061702940--hypothetical proteinCytoplasmic [Class 3]FALSO-H220707-054_F19_219DZAA037_premix.ab1617023099.083218111.9699999999999993e-108390plusPP_5408CDS6169113061702940--hypothetical proteinCytoplasmic [Class 3]FALSO-H220707-054_H21_219DZAA038_premix.ab1617054697.22200000000001108121.3e-45182minusPP_5409CDS6170466061723010-glmSL-glutamine/D-fructose-6-phosphate aminotransferase-FALSO-H220707-054_P19_219DZAA039_premix.ab1617054698.148108022.8e-47187minusPP_5409CDS6170466061723010-glmSL-glutamine/D-fructose-6-phosphate aminotransferase-FALSO-H220707-054_L19_219DZAA040_premix.ab1617054796.33109136.389999999999999e-44176minusPP_5409CDS6170466061723010-glmSL-glutamine/D-fructose-6-phosphate aminotransferase-FALSO-H220707-054_N21_219DZAA041_premix.ab1617054699.074108015.679999999999999e-49193minusPP_5409CDS6170466061723010-glmSL-glutamine/D-fructose-6-phosphate aminotransferase-FALSO-H220707-054_J19_219DZAA046_premix.ab1617053396.8420000000000195039.219999999999999e-38156minusPP_5409CDS6170466061723010-glmSL-glutamine/D-fructose-6-phosphate aminotransferase-FALSO-H220707-054_D21_219DZAA047_premix.ab1617023999.51700000000001207101.4899999999999996e-104377plusPP_5408CDS6169113061702940--hypothetical proteinCytoplasmic [Class 3]FALSO-