BLAST operations to/from an assembled genome (starting from raw reads)

Author

Menachem Sklarz

Affiliation

Bioinformatics Core Facility

Organization

National Institute of Biotechnology in the Negev, Ben Gurion University.

This workflow demonstrates various searching schemes with blast modules.

It begins with building an assembly from the reads, and then performs various operations on the assembly: (a) construction of a BLAST database from the assembly and searching the database with a fasta file supplied by the user; (b) annotation of the assembly and searching an external database with the predicted protein sequences and (c) searching for the predicted gene sequences in the assembly database.

Steps:

1. Merging and QC

2. Assembly using MEGAHIT (megahit modules at sample scope. i.e. one assembly per sample)

3. Constructing a BLAST db from the assembly using the makeblastdb module

4. Searching the assembly using a fasta query file (blast module).

5. Annotation of the assemblies using Prokka.

6. Using the resulting predicted gene sequences to search a BLAST database.

7. Using the alternative BLAST module (blast_new) to search the assembly for the predicted genes.

Workflow Schema

Requires

fastqc files. Either paired end or single end.

Programs required

• blast

• prokka

• megahit

• quast

• FastQC

• trimmomatic

• parse_blast

Example of Sample File

Title       Paired_end_project

#SampleID   Type    Path    lane
Sample1     Forward /path/to/Sample1_F1.fastq.gz 1
Sample1     Forward /path/to/Sample1_F2.fastq.gz 2
Sample1     Reverse /path/to/Sample1_R1.fastq.gz 1
Sample1     Reverse /path/to/Sample1_R2.fastq.gz 2
Sample2     Forward /path/to/Sample2_F1.fastq.gz 1
Sample2     Reverse /path/to/Sample2_R1.fastq.gz 1
Sample2     Forward /path/to/Sample2_F2.fastq.gz 2
Sample2     Reverse /path/to/Sample2_R2.fastq.gz 2

Download

The workflow file is available here