Assembling E. coli sequences with SPAdes

The goal of this tutorial is to show you the basics of assembly using the SPAdes assembler.

We’ll be using data from Efficient de novo assembly of single-cell bacterial genomes from short-read data sets, Chitsaz et al., 2011.

Packages to install

Download and insatll the SPAdes assembler:

cd ~/
tar xvfz SPAdes-3.6.0-Linux.tar.gz
export PATH=$PATH:$HOME/SPAdes-3.6.0-Linux/bin
echo 'export PATH=$PATH:$HOME/SPAdes-3.6.0-Linux/bin' >> ~/.bashrc

as well as Quast, software for evaluating the assembly against the known reference:

curl -L > quast-3.0.tar.gz
tar xvf quast-3.0.tar.gz

Getting the data

Now, let’s create a working directory:

cd /mnt
mkdir assembly
cd assembly

Copy in the E. coli data that you trimmed (Short read quality and trimming):

ln -fs /mnt/work/ecoli_ref-5m-trim.*.fq.gz .

Running an assembly

Now, let’s run an assembly: --12 -s -o spades.d

This will take about 15 minutes; it should end with te following output:

* Corrected reads are in /mnt/assembly/spades.d/corrected/
* Assembled contigs are in /mnt/assembly/spades.d/contigs.fasta (contigs.fastg)
* Assembled scaffolds are in /mnt/assembly/spades.d/scaffolds.fasta (scaffolds.fastg)

Looking at the assembly


~/quast-3.0/ spades.d/scaffolds.fasta -o report

and then look at the report:

less report/report.txt

You should see:

All statistics are based on contigs of size >= 500 bp, unless otherwise noted (e.g., "# contigs (>= 0 bp)" and "Total length (>= 0 bp)" include all contigs).

Assembly                   scaffolds
# contigs (>= 0 bp)        152
# contigs (>= 1000 bp)     80
Total length (>= 0 bp)     4571384
Total length (>= 1000 bp)  4551778
# contigs                  89
Largest contig             285527
Total length               4558170
GC (%)                     50.74
N50                        133088
N75                        67332
L50                        12
L75                        23
# N's per 100 kbp          0.00

What does this all mean?

Comparing and evaluating assemblies - QUAST

Download the true reference genome:

cd /mnt/assembly
curl -O
gunzip ecoliMG1655.fa.gz

and run QUAST again:

~/quast-3.0/ -R ecoliMG1655.fa spades.d/scaffolds.fasta -o report

Note that here we’re looking at all the assemblies we’ve generated.

Now look at the results:

less report/report.txt

and now we have a lot more information! What all is there?

Adding in Nanopore data and doing a hybrid assembly

One challenge with short read data like Illumina is that if there are repeats in the genome, they can’t be unambiguously resolved with short reads. Enter long reads, produced by PacBio and Nanopore sequencing. How much do long reads improve the assembly?

Let’s download some trial Nanopore data provided by Nick Loman:

cd /mnt/assembly
curl -O

Let’s take a quick look at these sequences and try BLASTing them at NCBI – note, you’ll want to use blastn, and choose “somewhat similar sequences” at the bottom. You can also restrict the BLAST search to E. coli MG1655.

Grab part of a sequence with gunzip -c FC20* | head and paste it into BLAST. What do you see?

Now, let’s try adding them into the assembly by running SPAdes with the Nanopore command line flag: --sc --12 -s --nanopore FC20.wf1.9.2D.pass.fasta.gz -o nanopore-ecoli-sc

How’d we do?

~/quast-3.0/ -R ecoliMG1655.fa nanopore-ecoli-sc/scaffolds.fasta -o n_report

Reference-free comparison

Above, we’ve been using the genome reference to do assembly comparisons – but often you don’t have one. What do you do to evaluate and compare assemblies without a reference?

One interesting trick is to just run QUAST with one assembly as a reference, and the other N assemblies against it. My only suggestion is to first eliminate short, fragmented contigs from the assembly you’re going to use as a reference.

Let’s try that, using from khmer: -l 1000 nanopore-ecoli-sc/scaffolds.fasta > spades-long.fa

and then re-run QUAST and put the output in report-noref/report.txt:

~/quast-3.0/ -R spades-long.fa spades.d/scaffolds.fasta \
         nanopore-ecoli-sc/scaffolds.fasta -o report-noref

When you look at the report,

less report-noref/report.txt

take particular note of the following –

Assembly                     spades.d_scaffolds  nanopore-ecoli-sc_scaffolds
# contigs (>= 0 bp)          152                 15
# contigs (>= 1000 bp)       80                  7
Total length (>= 0 bp)       4571384             4643870
Total length (>= 1000 bp)    4551778             4642289
# contigs                    89                  7
Largest contig               285527              3076878
Total length                 4558170             4642289
Reference length             4642289             4642289
Misassembled contigs length  134677              0
# local misassemblies        6                   0
Genome fraction (%)          98.161              99.923
Duplication ratio            1.000               1.001
# mismatches per 100 kbp     3.36                0.00

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