Short Read Assembly

Warning

These documents are not maintained and their instructions may be out of date. However the GED Lab does maintain the khmer protocols which may cover similar topics. See also the installation instructions for the current version of the khmer project.

Author:Jason Pell
Date:June 5, 2010
Last Updated:September 13, 2010

For this tutorial, we will be using the short read de novo assembler ABySS to assemble a bacterial genome.

Getting Started

Start or log in to an instance on Amazon EC2. If you haven’t already done so, make sure you get your instance updated. See: Installing your Amazon EC2 system.

Next, we need to obtain some different files. To obtain ABySS, use curl:

%% curl -O http://www.bcgsc.ca/downloads/abyss/abyss-1.2.3.tar.gz

We also need a library called SparseHash, which was created by Google. Obtain it by running:

%% curl -O http://google-sparsehash.googlecode.com/files/sparsehash-1.8.1.tar.gz

Now, we’ll grab our datasets:

%% curl -O ftp://ftp.ncbi.nlm.nih.gov/sra/SeqSamples/SRS000/SRS000537/SRX000430/SRR001666_1.fastq.bz2
%% curl -O ftp://ftp.ncbi.nlm.nih.gov/sra/SeqSamples/SRS000/SRS000537/SRX000430/SRR001666_2.fastq.bz2

This data is an Illumina run of E. coli with 36bp paired-end reads and a 500bp insert size. A similar dataset with an insert size of 200bp is available under accession number SRR001665 from NCBI if you are interested in trying to assemble them together.

Installing ABySS

Uncompress the tarballs and cd into the SparseHash directory:

%% tar xvzf sparsehash-1.8.1.tar.gz
%% tar xvzf abyss-1.2.3.tar.gz
%% cd sparsehash-1.8.1

Once you are in the SparseHash directory, run the following to install:

%% ./configure && make && make install

It should take a little while to finish. These three commands are pretty standard when you want to compile a UNIX package from source. The first command tunes the compilation and installation parameters for your particular system. Then, the package is compiled and installed on your system. Once it is finished, go to the ABySS directory and do the same thing:

%% cd ../abyss-1.2.3
%% ./configure --disable-openmp && make && make install

Running ABySS

Go back to your home directory. The assembly takes a while to run. To get around this, we’ll run the following:

%% mkdir k30
%% cd k30
%% time abyss-pe n=10 k=30 in='../SRR001666_*.fastq.bz2' name=ecoli > ecoli.out &

Let’s look at what is going on here. The time command simply outputs length of time that it took the assembly to run. The command abyss-pe is the paired-end option of the program. There are now a couple of different attributes that can be adjusted in order to give different results. The n parameter is the number of read pairs that must be found between two different contigs before the assembler considers attempting to scoffold or combine them. The k value represents the minimum overlap that is required between two reads for an overlap k-mer to be added to the de Bruijn graph.

The ampersand (&) at the end starts the job in the background so that you can log off and come back to it later. We are piping the output to ecoli.out so that you can look at the ABySS tool’s output on a typical run. The assembly took me about 47 minutes to complete on the smallest EC2 instance, so you can move on to installing screed and the assembly statistics Python script and take a look at the output later in the day.

Evaluating Assemblies

Assessing the quality of a de novo assembly where there is no good reference is still an open problem. However, there are many statistics that can be useful in comparing assemblies to one another. We have a Python script that provides many of these statistics; it’s in the ngs-course scripts, under ‘ngs-course/assembly/assemstats.py’.

Before you can run it, you must install screed, so we’ll run the following in the home directory:

%% apt-get install git-core
%% git clone git://github.com/acr/screed.git
%% cd screed
%% python setup.py install

Screed is a software package that was developed to easily parse and manipulate a large amount of short read sequences. Once the reads are indexed, you can very quickly retrieve any sequence you need by name. However, the assembly statistics Python script just uses the provided FASTA parser.

To obtain statistics on the file you just generated, go back to your home directory and run:

%% python ~/ngs-course/assembly/assemstats.py 0 k*/ecoli-contigs.fa

Other Stuff To Try

The optimal value for k depends greatly on the dataset. A lower value for k has greater sensitivity, but can produce more false overlaps. However, it is the best option when you don’t have high coverage. On the other hand, a high value for k will have a more accurate assembly and longer contigs, but you are likely to miss a lot of potential read overlaps, which means you need higher coverage to make up for the difference.

Try varying the value for k by creating a new directory for each value that you would like to test. Then, run:

%% python ~/ngs-course/assembly/assemstats.py 0 k*/ecoli-contigs.fa

again to see how the assemblies compare to each other. If you generate a lot of assemblies, you can copy and paste the output to a text file and import it into Excel as a space-delimited file.

Finally, if you have your own dataset, you can try to assemble it on your EC2 system. However, you may want to check memory usage with the top command in order to ensure that you are not using too much memory. If the ABySS memory usage is over 90%, you may be using virtual memory, which means the assembly will take much longer than necessary. See the next section for details on how to make your assemblies run faster with multiple cores.

Running ABySS in Parallel

One of the biggest advantages to using ABySS is that most of its different processes can be run in parallel. This means that you can split the work onto several different cores or even machines using OpenMPI. If you have a larger genome that you are interested in assembling, you will likely need to take advantage of this parallelism. To try this out, get a larger EC2 instance setup that has more than one core.

Obtain and install OpenMPI as follows:

%% curl -O http://www.open-mpi.org/software/ompi/v1.4/downloads/openmpi-1.4.2.tar.gz
%% tar xvzf openmpi-1.4.2.tar.gz
%% cd openmpi-1.4.2
%% ./configure && make all install
%% ldconfig

Now you will need to reconfigure and recompile the ABySS package to make use of OpenMPI:

%% cd ../abyss-1.2.3
%% ./configure && make && make install

Now you should be all set to take advantage of the multiple cores on the machine. Assuming that you have 2 cores available locally, we’ll make a new directory for another assembly and run the parallelized ABySS:

%% cd ~
%% mkdir k32
%% cd k32
%% time abyss-pe n=10 k=32 in='../SRR001666_*.fastq.bz2' name=ecoli np=2

Notice that the only change we made (other than using a different value of k) is the addition of the np=2 parameter that is passed. This tells OpenMPI that the work can be split up 2 different ways. You can get a lot more complicated by configuring OpenMPI to run across multiple machines that also have OpenMPI installed, but that is outside the scope of this tutorial.


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