Read mapping

Lecture 28
Unit 6
CSE 815 Bioinformatics
Hamid D. Ismail, Ph.D.
 Reads mapping/aligning
• Read mapping or sequence alignment is the process of aligning sequenced
DNA/RNA to a reference sequence.
• This reference could be a complete genome, a set of genes, or any other
DNA sequence.
• Most NGS applications depends on read mapping:
Reads
mapping

Genome Variant Gene Epigenetic Metagenomics

assembly calling expression analysis analysis

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 Understanding reads’ mapping
• Mapping is the process of finding the original location of a DNA read in a
reference sequence, typically a reference genome.

Reference sequences

Coverage
depth Aligned
short reads

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 Some factor affecting mapping quality
• Quality of reads: High-quality reads (i.e., those with few errors) are more
likely to map correctly to the reference sequence.
• Read length: Longer reads generally provide more reliable mapping
because they offer more sequence context to match against the reference
genome.
• Coverage depth refers to the number of times a particular base in a genome
is sequenced. This metric is essential for assessing the quality and reliability
of sequencing data.
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑏𝑎𝑠𝑒𝑠 𝑖𝑛 𝑟𝑒𝑎𝑑𝑠
𝐶𝑜𝑣𝑒𝑟𝑎𝑔𝑒 𝑑𝑒𝑝𝑡ℎ = × 100%
𝐺𝑒𝑛𝑜𝑚𝑒 𝑙𝑒𝑛𝑔𝑡ℎ
If the total number of bases is 30 million and the genome length is 3 million
bases, the average coverage depth would be 10X i.e. each base is
sequenced, on average, 10 times.

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 The steps of reads’ mapping
1- Acquiring the sequencing reads in fastq format (See unit 4)). For example;
download human WGS:
fasterq-dump --progress SRR26329589
2- Quality control of the sequencing reads to make sure that you fix the quality
problems (see unit 4). For example:
fasqc SRR26329589_1.fastq SRR26329589_2.fastq
3- Download the sequence of the reference genome of the organism.
4- Indexing the reference genome with samtools for faster and efficient alignment.
Different tools adopt different indexing methods.
5- Indexing the reference sequence and mapping reads using one of the read
aligners/ mappers.
6- Post-alignment processing (sorting, indexing, remove duplicates)
7- Quality assessment of alignment (coverage).

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 Downloading a reference genome
• Choose the database where you can download the reference genomes of the
organism under study.
• Most sequences of reference genomes are available in:
- NCBI genome database: https://www.ncbi.nlm.nih.gov/genome/
- Ensembl genome browser: https://useast.ensembl.org/index.html
- UCSC Genome Browser: https://genome.ucsc.edu/

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 Downloading a reference genome
• There are different way to download a reference genome from the NCBI
database:
1- NCBI datasets command line tool:
Download the reference genome sequence of:
"Severe acute respiratory syndrome coronavirus 2"

• This will download a compressed folder including the fasta sequence and
annotation files.

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 Downloading a reference genome
2- NCBI E-utilities or E-Direct if you know the accession number.

efetch -db nuccore \

-id ”NC_000001” \
-format fasta -mode text \
> NC_000001.fasta

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 Downloading a reference genome
3- Using BioPython

from Bio import Entrez

Entrez.email = "[email protected]"
handle = Entrez.efetch(db="nucleotide",
id="NC_000001",
rettype="fasta",
retmode="text")
data = handle.read()
handle.close()
with open("NC_000001.fasta", "w") as file:
file.write(data)

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 Downloading a reference genome
3- Using “wget” to download a reference genome from UCSC.
• The genomes are available at: http://hgdownload.soe.ucsc.edu/goldenPath/

wget http://hgdownload.soe.ucsc.edu/goldenPath/<GenomPath>

• Example: Downloading the human reference genome from UCSC.

• Be organized to create a directory for the reference genome (i.e. refHuman)
• Run the following command:

wget http://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz \
--no-check-certificate
Gunzip -d hg38.fa.gz

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 Indexing a reference genome with samtools
• The samtools is a collection of tools used for sequence indexing, and
mapped read manipulation.
• The “faidx” tool in samtools indexes a FASTA formatted sequence file and
creates an index file that allows for fast random access to the sequence data.
• The syntax for indexing a reference sequence:
samtools faidx your_reference_genome.fasta
• The output is a file with “.fai” extension, which includes indices of the fasta
file.
• The faidx index file is a text file consisting of lines, each with five TAB-delimited
columns:
▪ NAME (name of this reference sequence).
▪ LENGTH (length of sequence)
▪ OFFSET: Sequence's first base in bytes
▪ LINEBASES: the number of bases on each line
▪ LINEWIDTH: the number of bytes in each line

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 Indexing a reference genome with samtools
• For indexing the human reference genome:
samtools faidx hg38.fa

• The index file will be hg38.fa.fai

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 Read mapping with aligners
• Read aligners are tools used to align sequencing reads to a reference
genome.
• The reference genome is usually aligned by the aligner before used in the
alignment. Thus, mapping performed in two steps (i) indexing (ii) mapping.
• There are different data structures for indexing used by each aligner:
- Suffix Array: This is used by aligners like BWA.
- Burrows-Wheeler Transform (BWT) is used alongside suffix arrays to
compress the genome sequence to allows for efficient search operations.
- FM-index is based on the BWT, used by Bowtie, Bowtie2, and BWA.
- Hash Tables are used by aligners like STAR for quick lookup operations.

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 Read aligner data structures
Suffix BWT
array

Suffix Hash
tree table

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 Popular read aligners
• BWA (Burrows-Wheeler Aligner):
- BWA consists of three algorithms: BWA-backtrack, BWA-SW, and BWA-MEM.
- The BWA-MEM algorithm is particularly popular for high-quality alignments of
reads longer than 70bp and is efficient with high error rates.
• Bowtie2:
- Bowtie2 is a fast and memory-efficient tool for aligning sequencing reads to
long reference sequences.
- It is particularly good for aligning reads of about 50 up to 100s of characters to
relatively large genomes.
• STAR (Spliced Transcripts Alignment to a Reference):
- STAR is an aligner specifically designed for RNA-seq data, which can
efficiently handle spliced alignments of RNA-seq reads.
- It works well with reads from a wide range of lengths, (very short to very long).

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 Read mapping with BWA
• BWA (Burrows-Wheeler Aligner):
- BWA consists of three algorithms: BWA-backtrack, BWA-SW, and BWA-MEM.
- The BWA-MEM algorithm is particularly popular for high-quality alignments of
reads longer than 70bp and is efficient with high error rates.
• Bowtie2:
- Bowtie2 is a fast and memory-efficient tool for aligning sequencing reads to
long reference sequences.
- It is particularly good for aligning reads of about 50 up to 100s of characters to
relatively large genomes.
• STAR (Spliced Transcripts Alignment to a Reference):
- STAR is an aligner specifically designed for RNA-seq data, which can
efficiently handle spliced alignments of RNA-seq reads.
- It works well with reads from a wide range of lengths, (very short to very long).

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