Re-assembly

Overview

Teaching: 10 min
Exercises: 20 min

Questions

Objectives

• Run another assembly with the binned scaffolds.

• Check if the binned scaffolds are contained in any of the re-assembled scaffolds.

In this section we will try to get a complete genome from the scaffolds of our bin. For this, we will do another assembly as follows:

• Using the binned scaffolds as a backbone to guide the assembler
• Using only one sample to minimize between-sample heterogeneity and microorganisms’ diversity. Both factors make harder for the assembler to get a contiguous, complete genome.

Discussion: Sample for the re-assembly

Look at the heatmap in 3_profiles/heatmap.png and explain what you see. With which sample do you think it will be easier for the assembler to reconstruct the complete genome?

We will use SPAdes with the same parameters as in the cross-assembly, but also with --trusted-contigs for the binned scaffolds. This time it should take only one or two minutes.

# create a directory 4_re-assembly
$ mkdir 4_re-assembly

# change the extension of the FASTA file from .fna to .fasta . SPAdes complains otherwise
$ mv 0_raw-data/F2T1.fna 0_raw-data/F2T1.fasta

# run SPAdes
$ spades.py --iontorrent --only-assembler --trusted-contigs 3_profiles/scaffolds_corr_90.fasta --careful -s 0_raw-data/F2T1.fasta -o 4_re-assembly/spades_output

# inspect the results by looking at the re-assembled scaffolds identifiers
$ grep '>' 4_re-assembly/spades_output/scaffolds.fasta | head

To know if our binned scaffolds are contained in any scaffold of the re-assembly, we will use BLAST locally. The database will be the scaffolds from the re-assembly, and we will BLAST the binned scaffolds to them to see if there is any getting most of the matches. First we need to build the database with makeblastdb, and then do the actual BLAST with blastn.

# build the database
$ mv 4_re-assembly/spades_output/scaffolds.fasta 4_re-assembly/re-scaffolds.fasta
$ makeblastdb -in 4_re-assembly/re-scaffolds.fasta -out 4_re-assembly/re-scaffolds.blastdb -dbtype nucl

# run BLAST
$ blastn -db 4_re-assembly/re-scaffolds.blastdb -query 3_profiles/scaffolds_corr_90.fasta -out 4_re-assembly/corr_scaffolds_to_re.txt -outfmt 6

Open 4_re-assembly/corr_scaffolds_to_re.txt to inspect the results. Each line represents an alignment between a binned scaffold (or query, first column) and a re-assembled scaffold from the database (or subject, second column). Interesting columns to look at are the %similarity (column 3), alignment length (column 4), evalue (column 11) or bitscore (column 12).

If everything went well, you should see that one of re-assembled scaffolds is around ~96Kb and contains most of the scaffolds of our bin. Open the FASTA file, look for that scaffold and Blast it online.

Congratulations, you just rediscovered the crAssphage :)

Key Points

• If you got this far, you are a pro.