ViralBioinfAsia2022
7 Reference alignment
7.1 Overview
7.2 Practical data sets
Let’s start by doing some quick prep/housekeeping on our dataset:
unzip /home/manager/course_data/Reference_alignment/Reference_alignment.zip ———————————————————————– mv /home/manager/course_data/Reference_alignment/Reference_alignment/07-dengue_align /home/manager/course_data/Reference_alignment/ ———————————————————————– mv /home/manager/course_data/Reference_alignment/Reference_alignment/07-chikv-align /home/manager/course_data/Reference_alignment/ ———————————————————————– rm -r /home/manager/course_data/Reference_alignment/Reference_alignment/ ———————————————————————– rm -r /home/manager/course_data/Reference_alignment/__MACOSX/ ———————————————————————–
NOTE Be careful when using ‘rm -r’ ! You can easily delete quite a bit of data this way with no chance to recover it! When doing this, it’s a >good idea to use absolute paths to avoid deleting something important by accident.
Now that is done, let’s navigate to the proper folder:
cd /home/manager/course_data/Reference_alignment/07-dengue_align/ ———————————————————————–
7.3 Preparing our raw reads for mapping
Let’s first start by cleaning up our data:
trim_galore -q 25 –length 50 --paired dengue.read1.fq.gz dengue.read2.fq.gz ———————————————————————–
–q 25 = trim the 3’ end of the reads – remove nucleotides less than Phred Quality 25
--length 50 = after adapter and quality trimming, remove reads less than 50bp in length
--paired = the names of the paired fastq files to analyze in order (Read 1 then Read 2)
7.4 Aligning your reads to a reference genome with BWA
There are many tools available to align reads onto a reference sequence: BWA, Novoalign, bowtie2, STAR to name but a few. In this practical, we will be using BWA ([http://bio-bwa.sourceforge.net]).
BWA (Burrows–Wheeler aligner) is a commonly used software designed to map sequence reads to a reference genome. BWA actually has three variations: BWA-backtrack (Li and Durbin, 2009), BWA-SW (Li and Durbin, 2010) and BWA-MEM (Li, 2013). In this exercise, we will be using BWA-MEM, which is typically preferred for longer reads due to its speed and accuracy.
To get started, let’s start by indexing the reference genome:
bwa index dengue-genome.fa ———————————————————————–
You should now have some additional files in your directory:
-rw-r—– 1 manager manager 10B Aug 1 17:03 dengue-genome.fa.amb
-rw-r—– 1 manager manager 100B Aug 1 17:03 dengue-genome.fa.ann
-rw-r—– 1 manager manager 11K Aug 1 17:03 dengue-genome.fa.bwt
-rw-r—– 1 manager manager 2.6K Aug 1 17:03 dengue-genome.fa.pac
-rw-r—– 1 manager manager 5.3K Aug 1 17:03 dengue-genome.fa.sa
Now let’s align the samples to our reference genome using bwa mem. Alignment is just one single step with bwa mem:
bwa mem dengue-genome.fa dengue.read1.fq.gz dengue.read2.fq.gz > dengue-aln.sam ———————————————————————–
7.5 Manipulating your SAM file with SAMtools
SAMtools is a library and software package for parsing and manipulating alignments in the SAM/BAM format. It is a multifunctional set of tools that is able to convert from other alignment formats, sort and merge alignments, remove PCR duplicates, generate per-position information in the pileup format.
If you check your directory again, you should now see a file called dengue-aln.sam
This is an example of a “SAM” file, which is the common output file type for deep sequencing reads. SAM files tend to take up a lot of space on your computer however so let’s go ahead and convert this file to a “BAM” file, which is the compressed version. We will be using SAMtools to do this:
samtools view -bS dengue-aln.sam > dengue-aln.bam ———————————————————————–
In this command, we are telling SAMtools to view the file dengue-aln.sam and direct (>) the output into a file called dengue-aln.bam. The –bS tells samtools that we want the output in BAM format (b) and to auto-detect the format the input data is in (S).
To prepare our new .bam file for downstream use, we should now sort and index it using SAMtools:
samtools sort dengue-aln.bam -o dengue.bam ———————————————————————– samtools index dengue.bam ———————————————————————– rm dengue-aln.sam ———————————————————————– rm dengue-aln.bam ———————————————————————–
In this set of commands, we are using SAMtools to sort the BAM file dengue-aln.bam and output (-o) a new file called dengue.bam. We are then using samtools to index the BAM file dengue.bam - indexing enables faster searches downstream and requires the data to be sorted. Finally, since we no longer need our original uncompressed SAM file and unsorted BAM file and we don't want to tie up our server with unneeded files, we then use the rm command to remove the original dengue-aln.sam file and the old unsorted dengue-aln.bam file. Do be careful when using the rm command though, once you delete a file this way it is gone forever!
There should now be two additional files in your directory: dengue.bam (the BAM file) and dengue.bam.bai (the BAM index file). To check this and to view the sizes of the files you can use: ———————————————————————– ls -lh ———————————————————————– The -l modifier of this command will list your files in the long list format while the -h modifier will output the file sizes in a human readable format.
The output of this command should look something like this:
-rw-r----- 1 manager manager 18B Aug 1 18:28 annotation.txt
-rw-r----- 1 manager manager 11K Aug 1 16:55 dengue-genome.fa
-rw-r----- 1 manager manager 10B Aug 1 17:03 dengue-genome.fa.amb
-rw-r----- 1 manager manager 100B Aug 1 17:03 dengue-genome.fa.ann
-rw-r----- 1 manager manager 11K Aug 1 17:03 dengue-genome.fa.bwt
-rw-r----- 1 manager manager 26B Aug 1 19:27 dengue-genome.fa.fai
-rw-r----- 1 manager manager 2.6K Aug 1 17:03 dengue-genome.fa.pac
-rw-r----- 1 manager manager 5.3K Aug 1 17:03 dengue-genome.fa.sa
-rw-r----- 1 manager manager 424M Aug 1 17:22 dengue.bam
-rw-r----- 1 manager manager 96B Aug 1 17:23 dengue.bam.bai
-rw-r----- 1 manager manager 289M Aug 1 16:55 dengue.read1.fq.gz
-rw-r----- 1 manager manager 311M Aug 1 16:56 dengue.read2.fq.gz
One common thing to check is how many reads have aligned to the reference, and how many did not. Samtools can report this for us easily:
Number of mapped reads:
samtools view -c -F4 dengue.bam ———————————————————————–
An explanation of this command is as follows:
- samtools view to view the file dengue.bam
- –c = count the read alignments
- –F4 = skip read alignments that have the unmapped Flag 4
Number of unmapped reads:
samtools view -c -f4 dengue.bam ———————————————————————–
This time we use –f4 = only include read alignments that do have the unmapped flag 4
If your results show that you have 5,178,553 mapped reads and 1,740,506 unmapped reads, you are doing great!
Another way you can get these data is to use:
samtools idxstats dengue.bam ———————————————————————–
This should give you the mapped and unmapped data with a single command.
Finally, we can also dig deeper into the data to look at insert size length, number of mutations and overall coverage by creating a stats file:
samtools stats dengue.bam > dengue_stats.txt ———————————————————————–
The first section of this file is a summary of the aligned data set which can give you an idea of the quality of your data set and overall alignment. If you open the file you just made, you should be able to look through and find these numbers (among other things!):
SN raw total sequences: 6910050
SN last fragments: 3455025
SN reads mapped: 5169544
SN reads mapped and paired: 5036738
SN average length: 150
SN maximum length: 150
SN average quality: 35.1
SN insert size average: 241.1
SN insert size standard deviation: 70.9
7.6 Group practical
Now let’s take what you’ve learned and try it out on another dataset! In this example, we will be mapping reads to a Chikungunya virus genome.
To start, navigate to:
cd /home/manager/course_data/Reference_alignment/07-chikv-align/ ———————————————————————–
You should see four files:
annotation.txt
chikv-genome.fasta
chikv.read2.fq.gz
chikv.read1.fq.gz
Question 1: How many reads map to the Chikungunya genome?
Question 2: What is the insert size average?