ViralBioinfAsia2022
9 Consensus and variant calling theory and practical
9.1 Overview
9.2 Creating a consensus with BCFtools
Let’s start by preparing the folder and files we will be working with. Note: To make this run much faster on your laptops, we are going to be subsampling the .bam file you created in the ‘Reference_alignment’ module. When generating a consensus sequence on real data, I would strongly recommend using the full .bam file as the result may be more accurate. Here are the scripts you can copy and paste into your terminal:
mkdir /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected ———————————————————————– cp /home/manager/course_data/Reference_alignment/07-dengue_align/annotation.txt /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/ ———————————————————————– cp /home/manager/course_data/Reference_alignment/07-dengue_align/dengue-genome.fa /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/ ———————————————————————– samtools view -s 0.01 -b /home/manager/course_data/Reference_alignment/07-dengue_align/dengue.bam > /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/dengue-subsample_01p-aln.bam ———————————————————————– samtools sort /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/dengue-subsample_01p-aln.bam -o /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/dengue-subsample_01p.bam ———————————————————————– samtools index /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/dengue-subsample_01p.bam ———————————————————————– rm /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/dengue-subsample_01p-aln.bam ———————————————————————–
Finally, one of the programs we are going to use isn’t installed so let’s do that now:
sudo apt install bcftools ———————————————————————–
You’ll be using the ‘sudo’ (super user) command to install this so your system will ask you for a password. Type in your login (which should be ‘manager’)
Ok, all the prep work is done! Now let’s navigate to the proper folder so we can get started:
cd /home/manager/course_data/Consensus_and_variant_calling/dengue-corrected/ ———————————————————————–
Now that we have our alignment file, we can now generate a consensus sequence. We will explore two separate ways to generate this consensus. In the first example we will use bcftools mpileup and bcftools call to generate a “majority rules” consensus and the output format will be in .fasta file format. For many downstream applications this will be the output you will likely want. In the second example, we will also be using use bcftools mpileup and bcftools call but will also add in the vcfutils.pl vcf2fq script to generate a .fastq file that allows for ambiguities at each position if there is a mixture of variants present in the sample.
Example 1 - Using bcftools to generate a .fasta “majority rules” consensus file
We will be using the following options for bcftools mpileup and bcftools call respectively:
bcfools mpileup
-f, –fasta-ref FILE faidx indexed reference sequence file
bcftools call
-m, –multiallelic-caller alternative model for multiallelic and rare-variant calling (conflicts with -c)
-v, –variants-only output variant sites only
Here is how we will call this command:
samtools faidx dengue-genome.fa ———————————————————————– bcftools mpileup -f dengue-genome.fa dengue-subsample_01p.bam | bcftools call -mv -Oz –ploidy-file annotation.txt -o calls.vcf.gz ———————————————————————– tabix calls.vcf.gz ———————————————————————– cat dengue-genome.fa | bcftools consensus calls.vcf.gz > dengue-cns.fa ———————————————————————–
Finally, we will use a quick hack to rename the header of the .fasta file we just generated:
sed ‘s/>MN566112.1 Dengue virus 2 isolate New Caledonia-2018-AVS127, complete genome/>dengue-consensus-bcftools/’ dengue-cns.fa > dengue-consensus-bcftools.fa ———————————————————————– rm calls* ———————————————————————– rm dengue-cns.fa ———————————————————————–
We created (and removed) several files here. The one that contains the consensus genome you are looking for is dengue-consensus-bcftools.fa
Example 2 - Using samtools/bcftools along with vcfutils.pl vcf2fq to generate a .fastq consensus file that allows for ambiquities
Here we will be using the following options for samtools mpileup and bcftools call respectively:
bcftools mpileup
-f, --fasta-ref FILE faidx indexed reference sequence file
bcftools call
-c, --consensus-caller the original calling method (conflicts with -m)
Here's how we will call this command:
bcftools mpileup -f dengue-genome.fa dengue-subsample_01p.bam | bcftools call -c –ploidy-file annotation.txt | vcfutils.pl vcf2fq > dengue-consensus-full.fq ———————————————————————–
Finally, we will again use a quick hack to rename the header of your output file:
sed ‘s/@MN566112.1/>dengue-consensus-vcf2fq/’ dengue-consensus-full.fq > dengue-consensus-vcf2fq.fq ———————————————————————– rm dengue-consensus-full.fq ———————————————————————–
We created (and removed) several files here. The one that contains the consensus genome you are looking for is dengue-consensus-vcf2fq.fq
9.3 Visualizing the difference between these files
So what effectively is the difference between the dengue-consensus-bcftools.fa and dengue-consensus-vcf2fq.fq files we generated? If we were to align these back to the reference genome dengue-genome.fa we can see the ambiquity present in the dengue-consensus-vcf2fq.fq whereas the dengue-consensus-bcftools.fa merely shows a complete consensus change at this position.
9.4 Variant calling
There are a number of different variant callers available including FreeBayes, LoFreq, VarDict, and VarScan2. In this tutorial, we will be using LoFreq* (i.e. LoFreq version 2) - a fast and sensitive variant-caller for inferring SNVs and indels from next-generation sequencing data. LoFreq* makes full use of base-call qualities and other sources of errors inherent in sequencing (e.g. mapping or base/indel alignment uncertainty), which are usually ignored by other methods or only used for filtering.
LoFreq* can run on almost any type of aligned sequencing data (e.g. Illumina, IonTorrent or Pacbio) since no machine- or sequencing-technology dependent thresholds are used. It automatically adapts to changes in coverage and sequencing quality and can therefore be applied to a variety of data-sets e.g. viral/quasispecies, bacterial, metagenomics or somatic data.
LoFreq* is very sensitive; most notably, it is able to predict variants below the average base-call quality (i.e. sequencing error rate). Each variant call is assigned a p-value which allows for rigorous false positive control. Even though it uses no approximations or heuristics, it is very efficient due to several runtime optimizations and also provides a (pseudo-)parallel implementation. LoFreq* is generic and fast enough to be applied to high-coverage data and large genomes. On a single processor it takes a minute to analyze Dengue genome sequencing data with nearly 4000X coverage, roughly one hour to call SNVs on a 600X coverage E.coli genome and also roughly an hour to run on a 100X coverage human exome dataset.
For more details on the original version of LoFreq see Wilm et al. (2012).
LoFreq comes with a variety of subcommands. By just typing lofreq you will get a list of available commands. The command we will be using today is lofreq call which, will call variants in our dengue alignment from the previous section. The output for our variant calls will be in variant call format or, vcf.
9.5 Running LoFreq*
Now let’s see what lofreq call can do. For this tutorial, we will be using the standard presets for lofreq but when running this on your own datasets, I would encourage you to explore all the options available in lofreq by checking out the github page: https://github.com/andreas-wilm/lofreq3
lofreq call -b dengue-subsample_01p.bam -f dengue-genome.fa > dengue-variants-original-ref.vcf ———————————————————————–
This will take a few minutes to run depending on your system, but you should soon see a new file in your directory called: dengue-variants-original-ref.vcf
If you open this file with the less command, you should see that we have a large number of ‘consensus variants’ (i.e. variants with frequencies greater than 50%). This means that the reference we used here was not ideal. We can get better results if we re-map the reads to the new consensus genome we derived above (dengue-consensus-bcftools.fa) and then re-run lofreq:
bwa index dengue-consensus-bcftools.fa ———————————————————————– bwa mem dengue-consensus-bcftools.fa /home/manager/course_data/Reference_alignment/07-dengue_align/dengue.read1.fq.gz /home/manager/course_data/Reference_alignment/07-dengue_align/dengue.read2.fq.gz > dengue-consensus-aln.sam ———————————————————————– samtools view -bS dengue-consensus-aln.sam > dengue-consensus-aln.bam ———————————————————————– samtools sort dengue-consensus-aln.bam -o dengue-consensus.bam ———————————————————————– samtools index dengue-consensus.bam ———————————————————————– rm dengue-consensus-aln* ———————————————————————– samtools view -c -F4 dengue-consensus.bam ———————————————————————–
This final command should show that you have 5191658 reads mapped. Normally, I would suggest proceeding with your lofreq run from this point. In order to speed things up for the course, we are going to subsample our .bam file before we run lowfreq again:
samtools view -s 0.01 -b dengue-consensus.bam > dengue-consensus-subsample_01p-aln.bam ———————————————————————– samtools sort dengue-consensus-subsample_01p-aln.bam -o dengue-consensus-subsample_01p.bam ———————————————————————– samtools index dengue-consensus-subsample_01p.bam ———————————————————————– rm dengue-consensus-subsample_01p-aln.bam ———————————————————————– samtools view -c -F4 dengue-consensus-subsample_01p.bam ———————————————————————–
As you can see from this final command, we now have 51539 reads mapped - not ideal in a normal situation, but good enough for illustrative purposes.
Let’s start by building an index for our new consensus reference genome:
samtools faidx dengue-consensus-bcftools.fa ———————————————————————– lofreq call -b dengue-consensus-subsample_01p.bam -f dengue-consensus-bcftools.fa > dengue-variants-consensus-ref.vcf ———————————————————————–
This will take a few minutes to run depending on your system, but you should soon see a new file in your directory called: dengue-variants-consensus-ref.vcf
Done!
9.5 Practice
Now let’s take what you’ve learned and try it out on another dataset! In this example, we will be looking for variants from a Chikungunya virus alignment.
To start, let’s repeat the first set of instructions from above to prepare a new folder:
mkdir /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected ———————————————————————– cp /home/manager/course_data/Reference_alignment/07-chikv-align/annotation.txt /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/ ———————————————————————– cp /home/manager/course_data/Reference_alignment/07-chikv-align/chikv-genome.fasta /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/ ———————————————————————– samtools view -s 0.01 -b /home/manager/course_data/Reference_alignment/07-chikv-align/chikv.bam > /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/chikv-subsample_01p-aln.bam ———————————————————————– samtools sort /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/chikv-subsample_01p-aln.bam -o /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/chikv-subsample_01p.bam ———————————————————————– samtools index /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/chikv-subsample_01p.bam ———————————————————————– rm /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/chikv-subsample_01p-aln.bam ———————————————————————– cd /home/manager/course_data/Consensus_and_variant_calling/chikv-corrected/ ———————————————————————–
From here you should be able to follow the steps above to answer the questions below:
Question 1: How many mapped reads do you have in the 1% subsampled ‘chikv-consensus-subsample_01p.bam’ file?
Answer: 48194
Question 2: What is the allele frequency at position 757? Answer: A–>G AF=0.062893