12/12/22, 7:20 PM An Introduction to Genome Assembly

Galaxy Training!

An Introduction to Genome Assembly

Authors: Simon Gladman

Overview

 Questions:
How do we perform a very basic genome assembly from short read data?
 Objectives:
assemble some paired end reads using Velvet
examine the output of the assembly.
 Requirements:
Introduction to Galaxy Analyses
Sequence analysis
Quality Control:  slides -  hands-on
 Time estimation: 30 minutes
 Level: Introductory   
 Supporting Materials:
 Slides Datasets Workflows  Tours  FAQs  Recordings
 Available on these Galaxies
 Last modification: Oct 18, 2022
 License: Tutorial Content is licensed under Creative Commons Attribution 4.0 International License
The GTN Framework is licensed under MIT

Genome assembly with Velvet: Background

Get the data

Evaluate the input reads

Assemble reads with Velvet

Collect some statistics on the contigs

Discussion

Frequently Asked Questions

Feedback OPEN CHAT

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Citing this Tutorial

Genome assembly with Velvet:

Background
Velvet is one of a number of de novo assemblers that use short read sets as input (e.g. Illumina Reads).
The assembly method is based on the manipulation of de Bruijn graphs, via the removal of errors and
the simplification of repeated regions.

 Comment

For information about Velvet, you can check its (nice) Wikipedia page.

For this tutorial, we have a set of reads from an imaginary Staphylococcus aureus bacterium with a
miniature genome (197,394 bp). Our mutant strain read set was sequenced with the whole genome
shotgun method, using an Illumina DNA sequencing instrument. From these reads, we would like to
rebuild our imaginary Staphylococcus aureus bacterium via a de novo assembly of a short read set
using the Velvet assembler.

We also have a sequence for a reference genome that we will use later in the tutorial.

Agenda

In this tutorial, we will deal with:

1. Genome assembly with Velvet: Background
2. Get the data
3. Evaluate the input reads
4. Assemble reads with Velvet
5. Collect some statistics on the contigs
6. Discussion

Get the data

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1. Create and name a new history for this tutorial.

 Tip: Creating a new history 

2. Import from Zenodo or from the data library the files:

[Link]
[Link]
[Link]

 Tip: Importing data via links 

3. Change the name of the files to mutant_R1 , mutant_R2 and [Link] .

As a default, Galaxy uses the link as the name of the new dataset. It also does not link the
dataset to a database or a reference genome.

 Tip: Renaming a dataset 

4. Inspect the content of a file.

 Tip: Inspecting the content of a dataset 

 Question

1. What are four key features of a FASTQ file?

2. What is the main difference between a FASTQ and a FASTA file?

 Solution 

Link to here |  FAQs | Gitter Chat | Help Forum

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The reads have been sequenced from an imaginary Staphylococcus aureus bacterium using an Illumina
DNA sequencing instrument. We obtained the 2 files we imported ( mutant_R1 and mutant_R2 )

 Question

Why do we have 2 files here if we only sequenced the bacteria once?

 Solution 

Evaluate the input reads

Before doing any assembly, the first questions you should ask about your input reads include:

What is the coverage of my genome?

How good is my read set?
Do I need to ask for a new sequencing run?
Is it suitable for the analysis I need to do?

We will evaluate the input reads using the FastQC tool. This tool runs a standard series of tests on your
read set and returns a relatively easy-to-interpret report. We will use it to evaluate the quality of our
FASTQ files and combine the results with MultiQC.

 Hands-on: FastQC on a fastq file

1. FastQC  with the following parameters

 “Raw read data from your current history”: mutant_R1.fastq and
mutant_R2.fastq

 Tip: Select multiple datasets 

2. MultiQC  with the following parameters

“Software name”: FastQC
“Result file”: All raw data files generated by FastQC.
Link to here |  FAQs | Gitter Chat | Help Forum

MultiQC generates a webpage combining reports for FastQC on both datasets. It includes these graphs
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and tables:
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General statistics

This is important in setting maximum k-mer size for an assembly.

 Comment: Getting the length of sequences

Click on Configure Columns

Check Length
Close the window

 Question

1. How long are the sequences?

2. What is the average coverage of the genome, given our imaginary Staphylococcus
aureus bacterium has a genome of 197,394 bp?

 Solution 

Sequence Quality Histograms

Dips in quality near the beginning, middle or end of the reads may determine the
trimming/cleanup methods and parameters to be used, or may indicate technical problems with
the sequencing process/machine run.

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Figure 1: The mean quality value across each base position in the read

 Question

1. What does the y-axis represent?

2. Why is the quality score decreasing across the length of the reads?

 Solution 

Per Sequence GC Content

High GC organisms tend not to assemble well and may have an uneven read coverage
distribution.

Per Base N Content

The presence of large numbers of Ns in reads may point to a poor quality sequencing run. You
will need to trim these reads to remove Ns.

k-mer content

The presence of highly recurring k-mers may point to contamination of reads with barcodes or
adapter sequences.

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For a fuller discussion of FastQC outputs and warnings, see the FastQC website link, including the
section on each of the output reports, and examples of “good” and “bad” Illumina data.

We won’t be doing anything to these data to clean it up as there isn’t much need. Therefore we will
get on with the assembly!

Assemble reads with Velvet

Now, we want to assemble our reads to find the sequence of our imaginary Staphylococcus aureus
bacterium. We will perform a de novo assembly of the reads into long contiguous sequences using the
Velvet short read assembler.

The first step of the assembler is to build a de Bruijn graph. For that, it will break our reads into k-mers,
i.e. fragments of length k. Velvet requires the user to input a value of k (k-mer size) for the assembly
process. Small k-mers will give greater connectivity, but large k-mers will give better specificity.

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1. FASTQ interlacer  with the following parameters

“Type of paired-end datasets”: 2 separate datasets
“Left-hand mates”: mutant_R1.fastq
“Right-hand mates”: mutant_R2.fastq

Currently our paired-end reads are in 2 files (one with the forward reads and one with the
reverse reads), but Velvet requires only one file, where each read is next to its mate read. In
other words, if the reads are indexed from 0, then reads 0 and 1 are paired, 2 and 3, 4 and 5,
etc. Before doing the assembly per se, we need to prepare the files by combining them.

2. velveth  with the following parameters

“Hash Length”: 29
“Input Files”
Click on  “Input Files”
In “1: Input Files”
“file format”: fastq
“read type”: shortPaired reads
 “Dataset”: outputs of FASTQ interlacer

The tool takes our reads and break them into k-mers.

3. velvetg  with the following parameters

 “Velvet Dataset”: outputs of velveth
“Using Paired Reads”: Yes

This last tool actually does the assembly.

Link to here |  FAQs | Gitter Chat | Help Forum

Two files are generated:

A Contigs file

This file contains the sequences of the contigs longer than 2k. In the header of each contig, a bit
of information is added:

the k-mer length (called “length”): For the value of k chosen in the assembly, a measure of
how many k-mers overlap (by 1 bp each overlap) to give this length
the k-mer coverage (called “coverage”): For the value of k chosen in the assembly, a
measure of how many k-mers overlap each base position (in the assembly).

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A Stats file

This is a tabular file giving for each contig the k-mer lengths, k-mer coverages and other
measures.

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Collect some statistics on the contigs

 Question

1. How many contigs have been built?

2. What is the mean, min and max length of the contigs?

 Solution 

This table is limitted, but we will now collect more basic statistics on our assembly.

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1. Quast  with
“Contigs/scaffolds output file”: outputs of velvetg
“Type of assembly”: Genome
“Use a reference genome?”: Yes
“Reference genome”: [Link]
“Type of organism”: Prokaryotes
“Lower Threshold”: 500
“Comma-separated list of contig length thresholds”: 0,1000
Link to here |  FAQs | Gitter Chat | Help Forum

This tool generates 5 output files, but we will focus on the HTML report and the Icarus viewer.

 Question

1. What is represented in the Icarus viewer?

 Solution 

The HTML report reports many statistics computed by QUAST to assess the quality of the assembly:

Statistics about the quality of the assembly when compared to the reference (fraction of the
genome, duplication ratio, etc)
Misassembly statistics, including the number of misassemblies

A misassembly is a position in the contigs (breakpoints) that satisfy one of the following criteria:

the left flanking sequence aligns over 1 kbp away from the right flanking sequence on the
reference;
flanking sequences overlap on more than 1 kbp
flanking sequences align to different strands or different chromosomes
Unaligned regions in the assembly
Mismatches compared to the reference genomes
Statistics about the assembly per se, such as the number of contigs and the length of the largest
contig

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1. How many contigs have been constructed?

2. Which proportion of the reference genome do they represent?
3. How many misassemblies have been found?
4. Has the assembly introduced mismatches and indels?
5. What are N50 and L50?
6. Is there a bias in GC percentage induced by the assembly?

 Solution 

Discussion
 Hands-on: (Optional) Rerun for values k ranging from 31 to 101

1. velveth  with the same parameters as before except

“Hash Length”: a value between 31 and 101

2. velvetg  with the same parameters as before

3. Quast  with the same parameters as before
Link to here |  FAQs | Gitter Chat | Help Forum

We have completed an assembly on this data set for a number of k values ranging from 29 to 101. A
few of the assembly metrics appear below.

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Figure 2: Number of contigs in the assembly for various k-mer sizes

Figure 3: Largest contig in each of the assemblies by k-mer size

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Figure 4: Total number of base pairs in all the contigs for each assembly by k-mer size

Figure 5: N50 metric for each of the assemblies by k-mer size

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1. Are there any distinct features in the charts?

2. Does it look like one assembly might be better than some of the others?

The reasons for these patterns will be discussed in detail in the De Bruijn graph assembly slides and
tutorial.

 Key points

We assembled some Illumina fastq reads into contigs using a short read assembler called
Velvet

We showed what effect one of the key assembly parameters, the k-mer size, has on the
assembly

It looks as though there are some exploitable patterns in the metric data vs the k-mer size.

Frequently Asked Questions

Have questions about this tutorial? Check out the tutorial FAQ page or the FAQ page for the Assembly
topic to see if your question is listed there. If not, please ask your question on the GTN Gitter Channel
or the Galaxy Help Forum

Feedback
Did you use this material as an instructor? Feel free to give us feedback on how it went.
Did you use this material as a learner or student? Click the form below to leave feedback.

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Citing this Tutorial

1. Simon Gladman, An Introduction to Genome Assembly (Galaxy Training Materials).
[Link]
introduction/[Link] Online; accessed Mon Dec 12 2022
2. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems
10.1016/[Link].2018.05.012

 BibTeX 

 Congratulations on successfully completing this tutorial!

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