Alignment exercise

Overview

In this exercise you will practice aligning NGS data and working with alignment files.

1. Navigate to your home directory.
2. Create a directory called align.
3. Navigate to the align directory.

We will align two types of NGS data:

1. Pseudomonas single-end Illumina reads
2. Human paired-end Illumina reads

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P. aeruginosa single-end Illumina reads

Alignment using bwa mem

We will align single-end reads that have been trimmed from P. aeruginosa.

Raw data:

/home/projects/22126_NGS/exercises/alignment/SRR8002634_1.fastq.gz

Trimmed data:

/home/projects/22126_NGS/exercises/alignment/SRR8002634_1_trimmed.fq.gz

Reference genome:

/home/databases/references/P_aeruginosa/GCF_000006765.1_ASM676v1_genomic.fasta

The basic bwa mem command to align single-end reads is:

bwa mem [reference.fasta] [reads.fastq.gz] > [output.sam]

Remember: the > operator redirects standard output (STDOUT) to a file.

We have discussed multiplexing and read groups. It is good practice to add a read group ID and sample name during alignment. For example, if the read group is RG38 and the sample is SMPL96:

bwa mem -R "@RG\tID:RG38\tSM:SMPL96" [reference.fasta] [reads.fastq.gz] > [output.sam]

This information is crucial when you later merge multiple BAM files, so you can trace which reads came from which library or sample.

Task: Align the trimmed FASTQ file using the command above.

Q1: If you were not told which FASTQ file contains the trimmed reads, how could you determine it from the files themselves? (Hint: think of at least three different ways.)

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Inspecting the alignment

Assume you named your output file SRR8002634_1.sam. You can view it as:

less -S SRR8002634_1.sam

The -S option prevents line wrapping; press q to quit. Use the slides and the official SAM specification to interpret each field.

Answer the following:

Q2: How many lines does the header have (lines starting with @)?

Q3: What is the genomic coordinate (reference name and position) of the first read SRR8002634.1?

Q4: What is the mapping quality of the third read SRR8002634.3? What does that mapping quality tell you about this read?

Q5: Using the SAM flag definitions (see Picard flag explanation), determine among the first 8 reads how many map to the forward (+) strand and how many to the reverse (–) strand.

Q6: Is the 10th read SRR8002634.11 unmapped? (Note: SRR8002634.9 was removed by trimming, so numbering skips.) How did you determine this from the SAM fields?

To get basic alignment statistics, use:

samtools flagstat [input.sam]

Below is a brief explanation of the fields reported by flagstat:

Category	Meaning
mapQ	Mapping quality
QC-passed reads	Reads not marked as QC-failed; these are typically used for analysis.
QC-failed reads	Reads flagged as having problems by the processing pipeline; downstream tools usually ignore them.
total	Total number of alignments reported.
secondary	Additional alignments for reads that map equally well to multiple locations.
supplementary	Alignments for chimeric or split reads where different parts map to different locations.
duplicates	Reads marked as duplicates (e.g. PCR duplicates); will be discussed in the next class.
mapped	Number of reads with at least one reported alignment (not unmapped).
paired in sequencing	Reads that were sequenced as part of a pair (not single-end).
read1	First read in the pair (forward).
read2	Second read in the pair (reverse).
properly paired	Pairs that face each other and are within the expected insert size range.
with itself and mate mapped	Both the read and its mate are mapped (whether or not properly paired).
singletons	Reads that are mapped but whose mate is unmapped.
with mate mapped to a different chr	Reads whose mate is mapped to a different chromosome.

Q7: According to samtools flagstat, what fraction of reads did not align to the reference?

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Working with alignments

Format conversion

This should be the first and hopefully last time you work directly with SAM for large files.

First, check the SAM file size:

ls -lh SRR8002634_1.sam

Convert SAM to BAM:

samtools view -bS [input.sam] > [output.bam]

Check the BAM file size:

ls -lh SRR8002634_1.bam

-l gives a detailed listing (permissions, size, date). -h shows file sizes in human-readable form (e.g. 2.4M instead of 2469134 bytes).

The BAM file contains exactly the same alignments as the SAM file, but in binary form. To view it as SAM:

samtools view [input.bam] | less -S

You can filter reads based on SAM flags. For example, to include only unmapped reads:

samtools view -f 0x4 [input.bam]

To exclude unmapped reads:

samtools view -F 0x4 [input.bam]

The flag 0x4 corresponds to “read unmapped” (see the flag documentation).

Q8: What is the size ratio of SAM to BAM (SAM size divided by BAM size)?

Now convert BAM to CRAM, which compresses further using the reference:

samtools view -C -T [reference.fasta] [input.bam] > [output.cram]

Use the same reference FASTA you used for mapping. Check the CRAM file size with ls -lh.

To view CRAM as SAM:

samtools view -T [reference.fasta] [input.cram] | less -S

Q9: What is the size ratio of BAM to CRAM?

To save space, please remove the SAM and CRAM files (we will work with BAM only):

rm [file]

Sorting

Sort the BAM file by genomic coordinate:

samtools sort [input.bam] > [output.sorted.bam]

Be careful not to overwrite the original BAM file; for example:

• input.bam = SRR8002634_1.bam
• output.sorted.bam = SRR8002634_1.sorted.bam

Use samtools view and less -S to confirm that reads are ordered by reference and coordinate.

Index the sorted BAM file:

samtools index [input.sorted.bam]

Note: you cannot index an unsorted BAM file.

Retrieving a particular region

Once sorted and indexed, you can retrieve reads from a specific region:

samtools view [input.sorted.bam] [regionID]:[start]-[end]

For example, to get reads overlapping positions 1,000,000–1,000,100 on chromosome NC_002516.2:

samtools view SRR8002634_1.sorted.bam NC_002516.2:1000000-1000100

Q10: How many reads are aligned between positions 2,000,000 and 3,000,000 on the reference NC_002516.2?
Hint: do not save to a file; instead use:

samtools view [options] | wc -l

Q11: How many reads with mapping quality ≥ 30 are aligned between positions 2,000,000 and 3,000,000 on NC_002516.2?
Hint: run samtools view without arguments to see its options.

Average coverage

We will use mosdepth to measure the average coverage (mean number of reads covering each base in the genome):

/home/ctools/mosdepth/mosdepth [output_prefix] [input.sorted.bam]

Use any prefix you like (e.g. SRR8002634_1). mosdepth will write a summary file named [output_prefix].mosdepth.summary.txt.

Check the summary and see if the reported coverage makes sense given the data.

Q12: On average, how many reads cover a base in the genome? What is the maximum coverage (maximum number of reads covering a single position)?

You can also inspect per-position coverage using:

samtools mpileup [input.sorted.bam] | less -S

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The wrong reference genome?

Q13: Suppose you accidentally aligned the reads to a different bacterial reference genome, e.g. Yersinia pestis (the plague bacterium), a distant relative of Pseudomonas alcaligenes. Would the number of aligned reads go up or down compared to the correct reference? Why? What if the other species was very closely related — would you expect more or fewer reads to align?

Optional bonus: Try it.

Yersinia pestis reference:

/home/databases/references/Y_pestis/GCF_000222975.1_ASM22297v1_genomic.fasta

Pseudomonas alcaligenes reference:

/home/databases/references/P_alcaligenes/GCF_001597285.1_ASM159728v1_genomic.fasta

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Human paired-end Illumina reads

Aligning

We will align exome-seq reads from a Yoruba female.

Raw data:

/home/projects/22126_NGS/exercises/alignment/NA19201_1.fastq.gz
/home/projects/22126_NGS/exercises/alignment/NA19201_2.fastq.gz

NA19201_1.fastq.gz contains the forward reads; NA19201_2.fastq.gz contains the reverse reads. These reads are already trimmed.

Your goal is to write a single command line that:

1. Uses bwa mem to align the paired-end reads and produce SAM output.
2. Converts SAM to BAM.
3. Sorts the BAM file.

bwa mem syntax for paired-end reads:

bwa mem [reference.fasta] [forward.fastq.gz] [reverse.fastq.gz]

Human reference (GRCh38):

/home/databases/references/human/GRCh38_full_analysis_set_plus_decoy_hla.fa

Note: There are multiple versions of the human reference genome (e.g. hg18, hg19, hg38). Coordinates and even sequences can differ between versions. Always make sure to use the same reference version consistently in all steps of your analysis.

If possible, add a read group and sample name. For example, if read group is RG26 and sample is YRB42:

-R "@RG\tID:RG26\tSM:YRB42"

Converting and sorting:

• samtools view -bS [input.sam] converts SAM to BAM.
• When reading from STDIN, use /dev/stdin as the input.
• samtools sort [input.bam] sorts the BAM file.

Your combined command should:

• Run bwa mem,
• Pipe SAM output to samtools view,
• Pipe BAM output to samtools sort,
• Redirect the final sorted BAM to NA19201.bam.

The alignment may take around 10 minutes.

Q14: Write the full one-line command that performs alignment, SAM->BAM conversion, and sorting using pipes, and saves output as NA19201.bam.

Q15: What are two major advantages of using UNIX pipes instead of running each command separately and writing intermediate files?

Note: For speed, the provided reads only contain sequences mapping to chr20.

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Alignment statistics

flagstat

Q16: Using samtools flagstat, what proportion of reads aligned to the reference?

Q17: Using the same output, how many read pairs are marked as properly paired?

Q18: Index the BAM file, then run:

samtools view [input.bam] [chromosome]

Count how many reads align to chr20 (hint: pipe to wc -l). How many total reads are aligned to chr20?

stat

Generate additional alignment statistics using:

samtools stat [input.bam] > NA19201.stat

Generate plots from the statistics file:

plot-bamstats -p NA19201 NA19201.stat

Viewing the BAM statistics report:

The command above generates a set of PNG image files containing various BAM statistics (e.g. insert size, base composition, quality by cycle). The plots are created in the current directory.

If you are using MobaXterm, you can open the PNG files directly from the left-hand file panel.

If you are using macOS (or a standard terminal), copy the PNG files to your local computer and open them using any image viewer. For example:

scp stud0XX@pupilX.healthtech.dtu.dk:path/to/NA19201*.png .

Replace stud0XX with your student ID and pupilX with the compute node you are working on.

Q19: Look at the insert size distribution plot. What is the most common insert size (approximately)?

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Inspecting the alignment with samtools tview

We will use the text-based viewer samtools tview to inspect the human alignment around a potential variant.

First, make sure your BAM file is indexed:

samtools index NA19201.bam

Then start samtools tview:

samtools tview NA19201.bam /home/databases/references/human/GRCh38_full_analysis_set_plus_decoy_hla.fa

This opens the alignment in your terminal.

To jump to the region of interest (chr20:35,581,362):

1. Press g (for “goto”).
2. Type chr20:35581362 and press Enter.

You should now see:

• The reference sequence on the top line.
• Aligned reads below. Matching bases are often shown as . or ,, while mismatches are shown as the actual base (A/C/G/T).

Useful keys:

• ? – show help.
• Arrow keys – move left/right/up/down.
• q – quit tview.

Use this view to answer the following:

Q20: At position chr20:35,581,362, what bases are present in the sample reads?

Q21: How many reads support the non-consensus base at this position? (Count the reads showing the alternative base in tview.)

Q22: Based on the fraction of reads supporting the non-reference base, does this variant look more like a heterozygous or a homozygous variant? Explain briefly.

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Please find the answers here.

Congratulations, you finished the exercise!