Overview

SPAdes is a De Bruijn graph assembler which has become the preferred assembler in numerous labs and workflows. In this tutorial we will use SPAdes to assemble an E. coli genome from simulated Illumina reads. Genome assembly is quite difficult (though if Oxford Nanopore lowers its error rate assembly will likely get much easier and involve new tools). Genome assembly should only be used when you can not find a reference genome that is close to your own, if you are engaged in metagenomic projects where you don't know what organisms may be present, and in situations where you believe you may have novel sequence insertions into a genome of interest (Note that in this case however you would actually want to grab reads that do not map to your reference genome (and their pair in the case of paired end and mate-pair sequencing) rather than performing these functions on the fastq files you get from the raw sequencing.

Learning Objectives

• Run SPAdes to perform de novo assembly on fragment, paired-end, and mate-paired data.
• Use contig_stats.pl to display assembly statistics.
• Find proteins of interest in an assembly using Blast.

Installing SPAdes

Unfortunately, SPAdes does not exist as a module for loading on TACC nor is it available in the BioITeam materials. As it is available through the SPAdes website as binaries, is well supported, and doesn't require complex dependancies making it easy to install.

First, navigate to the SPAdes home page http://cab.spbu.ru/software/spades/ and download the linux binary distribution either directly to TACC using wget. While you could put the file anywhere on lonestar (and can easily move it around on lonestar with the mv command once it is there), I suggest downloading the file to a 'src' folder on $WORK as this is a good habit to get into. 

mkdir $WORK/src

Note that idev nodes have a tendency to download files from the internet much slower than the same file would download from the head node. If you are already in an idev node, it is likely faster to logout of the idev node (with the 'logout' command), execute the wget command listed below on the head node, and then start a new idev node after the download is complete.

cd $WORK/src
wget http://cab.spbu.ru/files/release3.13.0/SPAdes-3.13.0-Linux.tar.gz

Once the .tar.gz file has been placed in the $WORK/src folder using one of the above options, you need to extract the files.

cd $WORK/src
tar -xvzf SPAdes-3.13.0-Linux.tar.gz
# from the help file:
  # x = Extract
  # v = verbose
  # z = file is also gzipped
  # f = force 

Now that the files have been extracted you have a choice in how to use them: 1 option is to copy the binary files to a location that is already in your path (such as the $HOME/local/bin directory we set up for you in your .bashrc file), and the second option is to add the $WORK/src/SPAdes-3.13.0-Linux/bin folder to your path. This is a personal preference and I do not know how prevalent either choice is among other researchers. I know that my preference is to copy executable to known locations in the path rather than add a ton of different directories to my path, but others may feel differently. Below I present both options:

mkdir -p $HOME/local/bin $HOME/local/share # note deliberately creating 2 folders by using the space between them
cp $WORK/src/SPAdes-3.13.0-Linux/bin/* $HOME/local/bin  #Note that by specifying the full path all the files and the destination, this command can be run from anywhere on TACC.
cp -r $WORK/src/SPAdes-3.13.0-Linux/share/spades $HOME/local/share

export PATH=$WORK/src/SPAdes-3.13.0-Linux/bin:$PATH
# This line must be added to the .bashrc file found in your $HOME directory, in section 2. I typically add all modifications 1 after the other so the most recent thing I have added is the last line in this section, but is searched first when looking for commands.

If you have modified your PATH variable, you will need to log out of TACC and log back in before continuing.

Testing SPAdes installation

SPAdes comes with a self test option to make sure that the program is correctly installed. While this is not true of most programs, it is always a good idea to run whatever test data a program makes available rather than jumping straight into your own data as knowing there is an error in the program rather than your data makes troubleshooting very different. 

mkdir $SCRATCH/GVA_SPAdes_tutorial
cd $SCRATCH/GVA_SPAdes_tutorial
spades.py --test

Assuming everything goes correctly, there will be a large number of lines that pass pretty quickly with the last lines printed to the screen should being:

======= SPAdes pipeline finished.

========= TEST PASSED CORRECTLY.

SPAdes log can be found here: <$SCRATCH>/GVA_SPAdes_tutorial/spades_test/spades.log

Thank you for using SPAdes!

The lines immediately above this text list different output files and results from the assembly and will be true of all SPAdes runs and can be helpful for keeping track of where all your output ends up.

Set 1: Plasmid SPAdes

Assembling even small bacterial genomes can be incredibly time intensive (as well as memory intensive as highlighted above). Fortunately for this class, we can make use of the plasmid spades option to assemble and even smaller plasmid genome that is ~2000 bp long in only a few minutes. I analyzing this data on an idev node and then submitting the other data analysis for the bacterial genomes as a job to run overnight.

Data

Download the paired end fastq files which have had their adapters trimmed from the $BI/gva_course/Assembly/ directory.

cp $BI/gva_course/Assembly/*.fastq.gz $SCRATCH/GVA_SPAdes_tutorial
cd $SCRATCH/GVA_SPAdes_tutorial

SPAdes Assembly

Now let's use SPAdes to assemble the reads. As always its a good idea to get a look at what kind of options the program accepts using the -h option. SPAdes is actually written in python and the base script name is "spades.py". There are additional scripts that change many of the default options such as metaspades.py, plasmidspades.py, and rnaspades.py or these options can be set from the main spades.py script with the flags --meta, --plasmid, --rna respectively. For this tutorial lets use plasmidspades.py

Once you have figured out what options you need to use see if you can come up with a command to run on the single end and have the output go into a new directory called single_end using all 48 threads that are available (-t 48). The following command is expected to take less than 2 minutes.

plasmidspades.py -t 48 -o plasmid -1 SH1_1P.fastq.gz -2 SH1_2P.fastq.gz 

Evaluating the output

As you can see from listing the contents of the output 'plasmid' directory, there several new files generated. The 2 that I consider the most important are 1. contigs.fasta as this is the actual result of all the different contigs that were created. For circular chromosomes (such as plasmids) the goal would be that there is a single contig meaning that all of the reads were able to close the circle. The second useful file is spades.log as it has the information about the completed run that you can use to compare different samples or conditions in the event that you are interested trying to optimize the command options.

Looking at the contigs.fasta file can you answer the following questions (it is small enough to interrogate with cat or any other program)?

1.  How many contigs were generated?

   just 1 (its a fasta file so you focus on the > symbols to identify each different contig that is present)

   
   

   
   

2.  How how long is each the contig?

   2046bp

3.  How deep is the coverage of this plasmid?

   In this case the answer is ~180. This value can be particularly useful when you are trying to determine if novel DNA is present as a multi copy plasmid, or as something that has inserted into the chromosome. If it is inserted, you would expect the coverage to be similar to that of the chromosome, if it is a plasmid, it could be significantly higher.

Set 2: Whole Genome Simulated Data

Here we will look at 4 sets of data with library preparation conditions to evaluate how wet lab decisions influence outcomes on the computer

Data

mkdir  $SCRATCH/GVA_SPAdes_tutorial # you likely already did this when you ran the selftest
cp $BI/ngs_course/velvet/data/*/* $SCRATCH/GVA_SPAdes_tutorial
cd $SCRATCH/GVA_SPAdes_tutorial

Now we have a bunch of Illumina reads. These are simulated reads. If you'd ever like to simulate some on your own, you might try using Mason.

paired_end_2x100_ins_1500_c_20.fastq  paired_end_2x100_ins_400_c_20.fastq  single_end_100_c_50.fastq
paired_end_2x100_ins_3000_c_20.fastq  paired_end_2x100_ins_400_c_25.fastq
paired_end_2x100_ins_3000_c_25.fastq  paired_end_2x100_ins_400_c_50.fastq

There are 4 sets of simulated reads:

Note that these fastq files are "interleaved", with each read pair together one-after-the-other in the file. The #/1 and #/2 in the read names indicate the pairs. This is not something you will encounter very often if at all.

tacc:~$ head paired_end_2x100_ins_1500_c_20.fastq
@READ-1/1
TTTCACCGTTGACCAGCACCCAGTTCAGCGCCGCGCGACCACGATATTTTGGTAACAGCGAACCATGCAGATTAAATGCACCTGCGGGAGCGAGCTGCAA
+
*@A+<at:var at:name="55G" />T@@I&+@A+@@<at:var at:name="II" />G@+++A++GG++@++I@+@+G&/+I+GD+II@++G@@I?<at:var at:name="I" />@<at:var at:name="IIGGI" /><at:var at:name="A4" />6@A,+AT=<at:var at:name="G" />+@AA+GAG++@
@READ-1/2
TTAACACCGGGCTATAAGTACAATCTGACCGATATTAACGCCGCGATTGCCCTGACACAGTTAGTCAAATTAGAGCACCTCAACACCCGTCGGCGCGAAA
+
I@@H+A+@G+&+@AG+I>G+I@+CAIA++$+T<at:var at:name="GG" />@+++1+<at:var at:name="GI" />+ICI+A+@<at:var at:name="I" />++A+@@A.@<G@@+)GCGC%I@IIAA++++G+A;@+++@@@@6

Often your read pairs will be "separate" with the corresponding paired reads at the same index in two different files (each with exactly the same number of reads).

SPAdes Assembly

Now let's use SPAdes to assemble the reads. As always its a good idea to get a look at what kind of options the program accepts using the -h option. SPAdes is actually written in python and the base script name is "spades.py". There are additional scripts that change many of the default options such as metaspades.py, plasmidspades.py, and rnaspades.py or these options can be set from the main spades.py script with the flags --meta, --plasmid, --rna respectively.

Once you have figured out what options you need to use see if you can come up with a command to run on the single end and have the output go into a new directory called single_end using all 48 threads that are available (-t 48). The following command is expected to take between 60 and 70 minutes.

spades.py -t 48 -o single_end -s single_end_100_c_50.fastq 

If you are planning to run a job overnight, consider adding additional combinations of reads as individual commands to get an idea of how different insert sizes can play a role in final contig lengths. Just remember that insert length should always increase:

spades.py -t 48 -o 400_1500_3000 --12 paired_end_2x100_ins_400_c_50.fastq --12 paired_end_2x100_ins_1500_c_20.fastq --12 paired_end_2x100_ins_3000_c_25.fastq
spades.py -t 48 -o 400_and_1500 --12 paired_end_2x100_ins_400_c_50.fastq --12 paired_end_2x100_ins_1500_c_20.fastq
spades.py -t 48 -o 400_only --12 paired_end_2x100_ins_400_c_50.fastq 

Put all of the commands into a file named spades_commands. Be sure to ask for help if you are unsure how to use nano to do this.

Submitting the job

Once you have decided on the combinations you want to evaluate, use the 'wc -l' command to verify that your spades_commands file has as many commands as you expected, and then replace the ? in the following block with the output of that command

launcher_creator.py -N 4 -w 1 -n "spades" -t 06:00:00 -a "UT-2015-05-18" -j spades_commands -m "module load launcher/3.0.3" -q normal
sbatch spades.slurm

Evaluating the output

Explore each output directory that was created for each set of reads you interrogated. The actionable information is in the contigs.fasta file. The contig file is a fasta file ordered based on the length of the individual contig in decreasing order. The names of each individual contig lists the number of the contig (largest contig being named NODE_1 next largest being named NODE_2 and so on) followed by the length of the contig, and the coverage (labeled as cov on the line). Generally, the lower number of total contigs and the larger the length of each are regarded as better assemblies, but the number of chromosomes present in the organism is an important factor as well.

The grep command can be quite useful for isolating the names of the contigs with the information, especially when combined with the -c option to count the total number of contigs, or piping the reults to head/tail or both head and tail to isolate the top/bottom contigs.

# Count the total number of contigs:
grep -c "^>" single_end/contigs.fasta

# Determine the length of the 5 largest contigs:
grep "^>" single_end/contigs.fasta | head -n 5

# Determine the length of the 20 smallest contigs:
grep "^>" single_end/contigs.fasta | tail -n 20

# Determine the length of the 100th through 110th contigs:
grep "^>" single_end/contigs.fasta | head -n 110 | tail -n 10

If you ran multiple different combinations of reads for the simulated data how did the insert size effect the number of contigs? the length of the largest contigs? Why might larger insert sizes not help things very much?

The complete E. coli genome is about 4.6 Mb. Why weren't we able to assemble it, even with the "perfect" simulated data?

What comes next when working with your own data?

1. Look for things: If you're just after a few homologs, an operon, etc. you're probably done. Think about what question you are trying to answer. 
2. You can turn the contigs.fa into a blast database (formatdb or makeblastdb depending on which version of blast you have) or try multiple sequence alignments through NCBIs blast.
3. If you built your contigs based on a normal/control sample you can map other reads to the contigs using bowtie2 to try to identify variants in other samples.
4. If you don't think the contigs you have are "good enough"
   1. Try using Spades MismatchCorrector to see if you can improve the contigs you already have.
   2. Add additional sequencing libraries to try to connect some more contigs. Especially think about pacbio sequencing and oxford nanopore.

Original spades publication

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