SAMtools

Key takeaways

SAMtools belongs to computer science; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect SAMtools to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of SAMtools from memory before moving on to harder problems.

Reference excerpt

SAMtools is a set of utilities for interacting with and post-processing short DNA sequence read alignments in the SAM (Sequence Alignment/Map), BAM (Binary Alignment/Map) and CRAM formats, written by Heng Li. These files are generated as output by short read aligners like BWA. Both simple and advanced tools are provided, supporting complex tasks like variant calling and alignment viewing as well as sorting, indexing, data extraction and format conversion. SAM files can be very large (tens of Gigabytes is common), so compression is used to save space. SAM files are human-readable text files, and BAM files are simply their binary equivalent, whilst CRAM files are a restructured column-oriented binary container format. BAM files are typically compressed and more efficient for software to work with than SAM. SAMtools makes it possible to work directly with a compressed BAM file, without having to uncompress the whole file. Additionally, since the format for a SAM/BAM file is somewhat complex - containing reads, references, alignments, quality information, and user-specified annotations - SAMtools reduces the effort needed to use SAM/BAM files by hiding low-level details. As third-party projects were trying to use code from SAMtools despite it not being designed to be embedded in that way, the decision was taken in August 2014 to split the SAMtools package into a stand-alone software library with a well-defined API (HTSlib), a project for variant calling and manipulation of variant data (BCFtools), and the stand-alone SAMtools package for working with sequence alignment data.

Usage and commands Like many Unix commands, SAMtool commands follow a stream model, where data runs through each command as if carried on a conveyor belt. This allows combining multiple commands into a data processing pipeline. Although the final output can be very complex, only a limited number of simple commands are needed to produce it. If not specified, the standard streams (stdin, stdout, and stderr) are assumed. Data sent to stdout are printed to the screen by default but are easily redirected to another file using the normal Unix redirectors (> and >>), or to another command via a pipe (|).

SAMtools commands SAMtools provides the following commands, each invoked as samtools <subcommand>:

view The view command filters SAM or BAM formatted data. Using options and arguments it understands what data to select (possibly all of it) and passes only that data through. Input is usually a sam or bam file specified as an argument, but could be sam or bam data piped from any other command. Possible uses include extracting a subset of data into a new file, converting between BAM and SAM formats, and just looking at the raw file contents. The order of extracted reads is preserved. sort The sort command sorts a BAM file based on its position in the reference, as determined by its alignment. The element + coordinate in the reference that the first matched base in the read aligns to is used as the key to order it by. [TODO: verify]. The sorted output is dumped to a new file by default, although it can be directed to stdout (using the -o option). As sorting is memory intensive and BAM files can be large, this command supports a sectioning mode (with the -m options) to use at most a given amount of memory and generate multiple output file. These files can then be merged to produce a complete sorted BAM file [TODO - investigate the details of this more carefully]. index The index command creates a new index file that allows fast look-up of data in a (sorted) SAM or BAM. Like an index on a database, the generated *.sam.sai or *.bam.bai file allows programs that can read it to more efficiently work with the data in the associated files. tview The tview command starts an interactive ascii-based viewer that can be used to visualize how reads are aligned to specified small regions of the reference genome. Compared to a graphics based viewer like IGV, it has few features. Within the view, it is possible to jumping to different positions along reference elements (using 'g') and display help information ('?'). mpileup The mpileup command produces a pileup format (or BCF) file giving, for each genomic coordinate, the overlapping read bases and indels at that position in the input BAM files(s). This can be used for SNP calling for example. flagstat

Worked examples

Example 1 — a first encounter with SAMtools

Start with the simplest possible case. Write down what SAMtools claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In computer science, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to SAMtools before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about SAMtools ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of SAMtools

In research: SAMtools appears in computer science research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses SAMtools in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: SAMtools is common in secondary-school and first-year university syllabi. It links to neighbouring topics Bioinformatics algorithms, Bioinformatics software, DNA sequencing, so understanding it makes those chapters shorter.
In everyday life: Look for SAMtools outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

How to study SAMtools in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what SAMtools means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain SAMtools out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is SAMtools in simple terms?

SAMtools is a set of utilities for interacting with and post-processing short DNA sequence read alignments in the SAM (Sequence Alignment/Map), BAM (Binary Alignment/Map) and CRAM formats, written by Heng Li. These files are generated as output by short read aligners like BWA.

Why does SAMtools matter?

Because it connects several computer science ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study SAMtools?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on SAMtools.