- I have short reads and long reads. How do I assemble a genome?
- Perform Quality Control on your reads
- Perform a Small genome Assembly with Unicycler
- Evaluate the Quality of the Assembly with Quast
- Annotate the assembly with Prokka
- Introduction to Galaxy Analyses
- Sequence analysis
time Time estimation: 4 hours
The goal: E. coli C-1 assembly
In this tutorial we assemble and annotate the genome of E. coli strain C-1. This strain is routinely used in experimental evolution studies involving bacteriophages. For instance, now classic works by Holly Wichman and Jim Bull (Bull et al. 1997, Bull & Wichman 1998, Wichman et al. 1999) have been performed using this strain and bacteriophage phiX174.
To sequence the genome we have obtained the strain from the Yale E. coli Stock Center. The stock center sent us a filter paper disk infused with cells. The disk was placed in the center of an LB-agar plate. A single colony was picked and resuspended in a liquid LB medium, grown overnight, and genomic DNA was isolated. The DNA was then sequenced using two methods. To obtain high coverage, high accuracy data we used Illumina miSEQ to generated 250-bp paired end reads. To generate high length reads we used the Oxford Nanopore MinION machine.
Our goal is to reconstruct and annotate the full genome of E. coli C-1. As you will see in this tutorial a combination of many short, high accuracy reads with long, error-prone reads helps us produce an almost perfect assembly.
In this tutorial, we will deal with:
Background on data and tools
In this tutorial we will assemble a genome using two types of input data: (1) Illumina 250 bp paired-end reads and (2) Oxford Nanopore reads.
We generated 9,345,897 250 bp read pairs (library preparation performed on genomic DNA fragmented to mean size of 600 bp). However, to make sure that you can complete this tutorial in a finite amount of time we have downsampled (reduced in size) to 1,000,000 paired end reads - just enough to produce an accurate assembly.
Oxford Nanopore Data
There are 12,738 2d-reads. Maximum read length is 27,518 bp. The distribution of reads lengths looks like this:
You can see that there many reads under the second peak with median of approximately 7.5 kb.
warning Oxford Nanopore Data Format
Oxford Nanopore machines output data in fast5 format that contains additional information besides sequence data. In this tutorial we assume that this data is already converted into fastq. An additional tutorial dedicated to handling fast5 datasets will be developed shortly.
In this analysis we will perform two tasks: (1) assembly and (2) annotation. Below we will briefly outline the main ideas behind these two procedures and will describe the tools we will be using.
comment Knowing your assembly
Here we assume that you know a thing or two about assembly process. If you don’t: look at the slides accompanying this tutorial as well as other tutorials is this section.
For assembly we will be using Unicycler (also see publication by Wick:2017). Unicycler is designed specifically for hybrid assembly (that is, using both short- and long-read sequencing data) of small (e.g., bacterial, viral, organellar) genomes. In our hands it has produced complete high quality assemblies. Unicycler employs a multi-step process that utilizes a number of software tools:
Multisized deBruijn graph
Assemblers usually construct graphs for k-mers of a fixed size. We have noted that when k is small it is difficult to resolve the repeats. If k is too large a corresponding graph may be fragmented (especially if read coverage is low). SPAdes uses several values for k (that are either manually set or inferred automatically) to create a multisized graph that minimized tangledness and fragmentation by combining various k-mers (see Bankevich:2012):
Read pair utilization
While the use of paired reads and mate pairs is key to genome assembly, and not new, SPAdes utilizes so called paired DeBruin graphs to take the advantage of the paired end data. One of the key issues with paired DeBruin graphs is that the resulting genome assemblies do not tolerate variability in insert sizes: The initial formulation of paired DeBruijn graphs assumed constant distance between pairs of reads. In practice this distance is always variable. SPAdes performs k-bimer (these are k-mers derived from paired reads) adjustment to identify exact or nearly-exact distances for each k-bimer pair.
Sequencing data contains a substantial number of sequencing errors that manifest themselves as deviations (bulges and non-connected components) within the assembly graph. One way to improve the graph before assembly it is to minimize the number of sequencing errors by performing error correction. SPAdes uses BayesHammer to correct the reads. Here is a brief summary of what it does (see Nikolenko:2013):
- SPAdes (or rather BayesHammer) counts k-mers in reads and computes k-mer statistics that take into account base quality values.
- A Hamming graph is constructed in which k-mers are nodes. In this graph edges connect nodes (k-mers) if they differ from each other by a number of nucleotides up to a certain threshold (the Hamming distance). The graph is central to the error correction algorithm.
- Then Bayesian subclustering is done on the graph from the previous step. For each k-mer we now know the center of its subcluster.
- Solid k-mers are derived from cluster centers and are assumed to be error free.
- Solid k-mers are mapped back to the reads.
- Reads are corrected using solid k-mers:
In the case of the full dataset, SPAdes error correction changed 14,013,757 bases in 3,382,337 reads - a substantial fraction of the full ~18 million read dataset.
Pilon improves draft assemblies by using the information from the original reads aligned to the draft assembly. The following image from a publication by Walker:2014 highlights the steps of this process:
For annotation we are using Prokka (also see Seeman:2014). It scans the assembly generated with Unicycler with a set of feature prediction tools and compiles a list of genome annotation. It predicts the following features (Table from Seeman:2014):
|Feature||Tool used by Prokka|
|Protein-coding sequences (CDS)||Prodigal|
|Ribosomal RNA genes||RNAmmer|
|Transfer RNA genes||Aragorn|
|Signal leader peptides||SignalP|
|Non-coding RNA genes||Infernal|
Prokka predicts protein-coding regions using a two step process. It first identifies coordinates of putative genes using Prodigal and then compares the gene sequence against databases of known sequences at protein level using Blast+ and HMMer.
Let’s try it
Load data and assess quality
In this example we will use a downsampled version of E. coli C-1 Illumina and ONT sequencing data. These include 3 files: forward and reverse reads for Illumina, and Long read file produced by ONT. All data are in fastq format.
hands_on Hands-on: Load data into History
- To load data into your Galaxy instance:
- Log in into Galaxy
- Create new history (if you are new to Galaxy see Galaxy 101 tutorial first).
Get data tool as shown below (see these slides for an introduction on how to load data into Galaxy):
- Open Zenodo link in a new browser window and right-click on dataset links:
And paste them into the Galaxy upload:
If all goes well you will see datasets uploading and changing states from gray to green as shown below. The figure below also shows how datasets can be tagged.
Assess Read Quality
hands_on Hands-on: Quality Control
- param-files “Short read data from your current history”: Select all three FastQ datasets simultaneously
MultiQC tool: to generate a summary of the FastQC reports with
- “Which tool was used generate logs?”:
- “Type of FastQC output”:
- param-files “FastQC Output”:
RawDataoutputs of FastQC
A quick look at quality score distribution will show a confusing picture:
So let’s zoom in into Illumina data:
Assembly with Unicycler
Now it is time to perform assembly.
hands_on Hands-on: Unicycler Assembly
- Unicycler tool with the following parameters :
- “Paired or Single end data?” to
- “First Set of reads” to the forward reads file
- “Second Set of reads” to the reverse reads file
- “Long reads” to the MinION file
- Use default parameters
Assembly takes time!
There is no such thing as Assembly in real time. It takes time so it is a good time to have lunch or at least coffee. This Unicycler run will take anywhere between 90 minutes and two hours.
Assess Assembly quality with Quast
Quast is a tool providing quality metrics for assemblies, and can also be used to compare multiple assemblies. The tool can also take an optional reference file as input, and will provide complementary metrics.
hands_on Hands-on: Assembly Quality
- Quast tool: with the following parameters
- “Contigs/scaffolds output file”: Select the fasta file resulting from the Unicycler assembly.
The Quast tool outputs assembly metrics as an html file with metrics and graphs. The image below looks exceptionally boring. This is a good thing:
One can see that there are two (!) contigs. The largest contig is 4,576,290 bp (for comparison E. coli K12 MG1655 strain genome length is 4,656,144 bp) and the smallest is 4,581,676 (total length) - 4,576,290 (length of the largest) = 5,386 bp. When we analyzed this dataset for the first time we were initially puzzled by this second contig. But we quickly realized that this is simply the genome of bacteriophage phiX174 which is routinely used as a spike-in in Illumina sequencing. Thus we have two genomes: the one of E.coli C-1 and phiX174! We can now use Prokka to annotate our two genomes.
Annotation with Prokka
hands_on Hands-on: Annotation
- Prokka tool:
- “Contigs to annotate”: Select the assembly ouput of
- “Genus name”:
- “Species name”:
- “Strain name”:
- “Use genus-specific BLAST database”:
Prokka outputs 10 datasets (including two log files). These are in various formats:
- txt : Provides Statistics on the annotation : number of CDS predicted, number of rRNA etc.
- tbl : Provides a tabulated list of annotated features.
- fsa : Nucleotide fasta file of the input contig sequence.
- sqn : ASN.1 format file for submission to GenBank.
- ffn : Nucleotide FASTA file of all the prediction transcripts.
- faa : Protein FASTA file of the translated CDS sequences.
- fna : Nucleotide fasta file of the input contig sequence.
- gbk : GenBank file.
- gff : gff3 file.
Visualize the results in IGV
Let’s look at the entire assembly and its annotation in the genome browser. We can do this using Integrated Genome Browser (IGV).
Visualization requires a local installation of IGV. If you have IGV installed - just start it. If you don’t - read on.
Go to IGV download page and select one of the options. The one I would try first would be Java Web Start. Simply click the Launch button for 10 GB distribution.
hands_on Hands-on: Visualization in IGV
Start IGV. It will look something like this:
Locate the output of Unicycler and expand it :
Click on the local link highlighted with orange outline. The browser will change:
Let’s add Prokka annotations to the browser image. For this simply expand Prokka’s GFF3 dataset and click on the local link:
You will now see the annotations within the browser window:
keypoints Key points
- We learned about the strategies used by assemblers for hybrid assemblies
- We performed an hybrid assembly of a bacterial genome and its annotation
- Unicycler is a pipeline bases on Spades and Pilon dedicated to hybrid assembly of Small genomes
- Combination of short and long reads helped us produce an almost perfect assembly
Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.
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