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SAM Input and Output in SeqAn

Learning Objective:

Learning Objective:
You will get an overview of how to read and write SAM/BAM files. This tutorial is a walk-through with links into the API documentation and is also meant as a source for copy-and-paste code.

Difficulty | Medium
Duration | 60 min
Prerequisite tutorials | Quick Setup (using CMake), Alphabets in SeqAn, Sequence File Input and Output
Recommended reading |

# Introduction

SAM files are used to store pairwise alignments between two (biological) sequences. There are also other output formats, like BLAST, which can store sequence alignments, but in this tutorial we will focus on SAM/BAM files. In addition to the alignment, these formats store information such as the start positions or mapping qualities. SAM files are a little more complex than sequence files, but the basic design is the same. If you are new to SeqAn, we strongly recommend completing the tutorial Sequence File Input and Output first.

# SAM/BAM file formats

## SAM format

SAM stands for Sequence Alignment/Map format. It is a TAB-delimited text format consisting of a header section, which is optional, and an alignment section (see the official SAM specifications).

Here is an example of a SAM file:

@HD VN:1.6 SO:coordinate
@SQ SN:ref LN:45
r001 99 ref 7 30 8M2I4M1D3M = 37 39 TTAGATAAAGGATACTG *
r003 0 ref 9 30 5S6M * 0 0 GCCTAAGCTAA *
r004 0 ref 16 30 6M14N5M * 0 0 ATAGCTTCAGC *
r003 2064 ref 29 17 5M * 0 0 TAGGC *
r001 147 ref 37 30 9M = 7 -39 CAGCGGCAT * NM:i:1

The following table summarises the columns of a SAM file:

# | SAM Column ID | Description
--- | --- | ---
1 | QNAME | Query template NAME
2 | FLAG | bitwise FLAG
3 | RNAME | Reference sequence NAME
4 | POS | 1-based leftmost mapping POSition
5 | MAPQ | MAPping Quality
6 | CIGAR | CIGAR string
7 | RNEXT | Reference name of the mate/next read
8 | PNEXT | Position of the mate/next read
9 | TLEN | observed Template LENgth
10 | SEQ | segment SEQuence
11 | QUAL | ASCII of Phred-scaled base QUALity+33

If you want to read more about the SAM format, take a look at the official specifications.

## BAM format

BAM is the binary format version of SAM. It provides the same data as the SAM format with negligible and subtle differences in most use cases.

# SAM file fields

To make things clearer, here is the table of SAM columns and the corresponding fields of a SAM file record:

# | SAM Column ID | FIELD name | seqan3::field
--- | --- | --- | ---
1 | QNAME | seqan3::sam_record::id | seqan3::field::id
2 | FLAG | seqan3::sam_record::flag | seqan3::field::flag
3 | RNAME | seqan3::sam_record::reference_id | seqan3::field::ref_id
4 | POS | seqan3::sam_record::reference_position | seqan3::field::ref_offset
5 | MAPQ | seqan3::sam_record::mapping_quality | seqan3::field::mapq
6 | CIGAR | seqan3::sam_record::cigar_sequence | seqan3::field::cigar
7 | RNEXT | seqan3::sam_record::mate_reference_id | seqan3::field::mate
8 | PNEXT | seqan3::sam_record::mate_position | seqan3::field::mate
9 | TLEN | seqan3::sam_record::template_length | seqan3::field::mate
10 | SEQ | seqan3::sam_record::sequence | seqan3::field::seq
11 | QUAL | seqan3::sam_record::base_qualities | seqan3::field::qual

SAM files provide following additional fields:

- seqan3::sam_record::tags (seqan3::field::tags)
- seqan3::sam_record::header_ptr (seqan3::field::header_ptr)

## File extensions

The formerly introduced formats can be identified by the following file name extensions (this is important for automatic format detection from a file name as you will learn in the next section).

File Format | File Extensions
--- | ---
SAM | .sam
BAM | .bam

You can access and modify the valid file extensions via the file_extension member variable in a format tag:

#include <seqan3/core/debug_stream.hpp>
#include <seqan3/io/sam_file/all.hpp>
int main()
{
seqan3::debug_stream << seqan3::format_sam::file_extensions << '\n'; // prints [sam]
seqan3::format_sam::file_extensions.push_back("sm");
seqan3::debug_stream << seqan3::format_sam::file_extensions << '\n'; // prints [sam,sm]
}

# Reading SAM files

Before we start, you should copy and paste this example file into a file location of your choice (we use the current path in the examples, so make sure you adjust your path).

Attention
  Make sure the file you copied is tab delimited!

## Construction

The construction works analogously to sequence files by passing a file name, in which case, all template parameters are automatically deduced (by the file name extension). Or you can pass a stream (e.g. std::cin or std::stringstream), but then you need to know your format beforehand:

#include <filesystem>
#include <seqan3/io/sam_file/all.hpp>
int main()
{
auto filename = std::filesystem::current_path() / "my.sam";
seqan3::sam_file_input fin_from_filename{filename};
seqan3::sam_file_input fin_from_stream{std::cin, seqan3::format_sam{}};
return 0;
}

## Accessing individual record members

You can access a record member like this:

#include <seqan3/io/sam_file/all.hpp>
int main()
{
auto filename = std::filesystem::current_path() / "example.sam";
seqan3::sam_file_input fin{filename};
// for (seqan3::sam_record record : fin) // this will copy the record
for (auto && record : fin) // this will pass the record by reference (no copy)
{
seqan3::debug_stream << record.id() << '\n';
seqan3::debug_stream << record.sequence() << '\n';
seqan3::debug_stream << record.flag() << '\n' << '\n';
}
}

See seqan3::sam_record for all data accessors.

#### Assignment 1: Accumulating mapping qualities

Let's assume we want to compute the average mapping quality of a SAM file.

For this purpose, write a small program that

- only reads the mapping quality (seqan3::sam_record::mapping_quality) of a SAM file and
- computes the average of all qualities.

Use the following file to test your program:

@HD VN:1.6 SO:coordinate
@SQ SN:ref LN:45
r001 99 ref 7 30 8M2I4M1D3M = 37 39 TTAGATAAAGGATACTG *
r003 0 ref 9 30 5S6M * 0 0 GCCTAAGCTAA * SA:Z:ref,29,-,6H5M,17,0;
r004 0 ref 16 30 6M14N5M * 0 0 ATAGCTTCAGC *
r003 2064 ref 29 17 5M * 0 0 TAGGC * SA:Z:ref,9,+,5S6M,30,1;
r001 147 ref 37 30 9M = 7 -39 CAGCGGCAT * NM:i:1

It should output:

Average: 27.4

Solution
#include <filesystem>
#include <ranges>
#include <seqan3/core/debug_stream.hpp>
#include <seqan3/io/sam_file/all.hpp>
int main()
{
auto filename = std::filesystem::current_path() / "my.sam";
seqan3::sam_file_input fin{filename};
double sum{};
size_t count{};
std::ranges::for_each(fin,
[&sum, &count](auto & record)
{
sum += record.mapping_quality();
++count;
});
seqan3::debug_stream << "Average: " << (sum / count) << '\n';
}

# Alignment representation in SAM/BAM files

The SAM format is the common output format of read mappers, where you align short read sequences to one or more large reference sequences. In fact, the SAM format stores those alignment information only partially: It does not store the reference sequence, but only the query/read sequence and a CIGAR string representing the alignment based on the read.

Take this SAM record as an example:

r003 73 ref 3 17 1M1D4M * 0 0 TAGGC *

The record gives you the following information: A read with name r003 has been mapped to a reference with name ref at position 3 (in the reference, counting from 1) with a quality of 17 (Phred scaled). The flag has a value of 73 which indicates that the read is paired, the first in the pair, but the mate is unmapped (see this website for a nice explanation of SAM flags). Fields set to 0 or * indicate empty fields and contain no valuable information.

The cigar string is 1M1D4M which represents the following alignment:

1 2 3 4 5 6 7 8 9 ...
ref N N N N N N N N N ...
read T - A G G C

where the reference sequence is not known (represented by N). You will learn in the next section how to handle additional reference sequence information.

If you want to read up more about cigar strings, take a look at the SAM specifications or the SAMtools paper.

## Reading the CIGAR string

By default, the seqan3::sam_file_input will always read the seqan3::sam_record::cigar_sequence and store it into a std::vector<seqan3::cigar>:

#include <filesystem>
#include <seqan3/core/debug_stream.hpp>
#include <seqan3/io/sam_file/all.hpp>
int main()
{
auto filename = std::filesystem::current_path() / "my.sam";
seqan3::sam_file_input fin{filename}; // default fields
for (auto & record : fin)
seqan3::debug_stream << record.cigar_sequence() << '\n'; // access cigar vector
}

## Transforming the CIGAR information into an alignment

In SeqAn, we represent an alignment as a tuple of two seqan3::aligned_sequences.

The conversion from a CIGAR string to an alignment can be done with the function seqan3::alignment_from_cigar. You need to pass the reference sequence with the position the read was aligned to and the read sequence:

// SPDX-FileCopyrightText: 2006-2025 Knut Reinert & Freie Universität Berlin
// SPDX-FileCopyrightText: 2016-2025 Knut Reinert & MPI für molekulare Genetik
// SPDX-License-Identifier: CC0-1.0
#include <seqan3/alignment/cigar_conversion/alignment_from_cigar.hpp>
#include <seqan3/core/debug_stream.hpp>
#include <seqan3/io/sam_file/all.hpp>
using namespace seqan3::literals;
auto sam_file_raw = R"(@HD VN:1.6
@SQ SN:ref LN:34
read1 41 ref 1 61 1S1M1D1M1I ref 10 300 ACGT !##$ AS:i:2 NM:i:7
read2 42 ref 2 62 1H7M1D1M1S2H ref 10 300 AGGCTGNAG !##$&'()* xy:B:S,3,4,5
read3 43 ref 3 63 1S1M1P1M1I1M1I1D1M1S ref 10 300 GGAGTATA !!*+,-./
)";
int main()
{
// The reference sequence might be read from a different file.
seqan3::dna5_vector reference = "ACTGATCGAGAGGATCTAGAGGAGATCGTAGGAC"_dna5;
seqan3::sam_file_input fin{std::istringstream{sam_file_raw}, seqan3::format_sam{}};
// You will probably read it from a file, e.g., like this:
// seqan3::sam_file_input fin{"test.sam"};
for (auto && rec : fin)
{
auto alignment =
alignment_from_cigar(rec.cigar_sequence(), reference, rec.reference_position().value(), rec.sequence());
seqan3::debug_stream << alignment << '\n';
}
// prints:
// (ACT-,C-GT)
// (CTGATCGAG,AGGCTGN-A)
// (T-G-A-TC,G-AGTA-T)
}
alignment_from_cigar.hpp
Provides the function seqan3::alignment_from_cigar.
std::istringstream
seqan3::format_sam
The SAM format (tag).
Definition format_sam.hpp:105
seqan3::sam_file_input
A class for reading SAM files, both SAM and its binary representation BAM are supported.
Definition sam_file/input.hpp:236
debug_stream.hpp
Provides seqan3::debug_stream and related types.
seqan3::alignment_from_cigar
auto alignment_from_cigar(std::vector< cigar > const &cigar_vector, reference_type const &reference, uint32_t const zero_based_reference_start_position, sequence_type const &query)
Construct an alignment from a CIGAR string and the corresponding sequences.
Definition alignment_from_cigar.hpp:81
seqan3::debug_stream
debug_stream_type debug_stream
A global instance of seqan3::debug_stream_type.
Definition debug_stream.hpp:38
seqan3::field::alignment
@ alignment
The (pairwise) alignment stored in an object that models seqan3::detail::pairwise_alignment.
Definition record.hpp:78
all.hpp
Meta-header for the IO / SAM File submodule .
seqan3::literals
The SeqAn namespace for literals.

The code will print the following:

0
ACT-
C-GT
0 .
CTGATCGAG
| |
AGGCTGN-A
0 .
T-G-A-TC
|
G-AGTA-T

#### Assignment 2: Combining sequence and alignment files

Read the following reference sequence FASTA file (see the sequence file tutorial if you need a reminder):

>chr1
ACAGCAGGCATCTATCGGCGGATCGATCAGGCAGGCAGCTACTGG
>chr2
ACAGCAGGCATCTATCGGCGGATCGATCAGGCAGGCAGCTACTGTAATGGCATCAAAATCGGCATG

Then read the following SAM file while providing the reference sequence information.

@HD VN:1.6 SO:coordinate
@SQ SN:chr1 LN:45
@SQ SN:chr2 LN:66
r001 99 chr1 7 60 8M2I4M1D3M = 37 39 TTAGATAAAGGATACTG *
r003 0 chr1 9 60 5S6M * 0 0 GCCTAAGCTAA *
r004 0 chr2 16 60 6M14N5M * 0 0 ATAGCTTCAGC *
r003 2064 chr2 18 10 5M * 0 0 TAGGC *

Only use

- seqan3::sam_record::id,
- seqan3::sam_record::reference_id,
- seqan3::sam_record::mapping_quality, and
- seqan3::sam_record::cigar.

With that information, do the following:

- Filter the alignment records and only take those with a mapping quality >= 30. (Take a look at the tutorial Reading paired-end reads for a reminder about using views on files)
- For the resulting alignments, print which read was mapped against which reference id and the number of seqan3::gaps in each sequence (aligned reference and read sequence).

Hint
Given your reference sequences, you need to know which reference sequence to use for the alignment. For this purpose, you can access record.reference_id() which gives you the position of the respective reference sequence.

Your program should print the following:

r001 mapped against 0 with 1 gaps in the read sequence and 2 gaps in the reference sequence.
r003 mapped against 0 with 0 gaps in the read sequence and 0 gaps in the reference sequence.
r004 mapped against 1 with 14 gaps in the read sequence and 0 gaps in the reference sequence.

Solution
#include <algorithm> // std::ranges::count
#include <filesystem>
#include <ranges>
#include <string>
#include <vector>
#include <seqan3/alignment/cigar_conversion/alignment_from_cigar.hpp>
#include <seqan3/alphabet/gap/gap.hpp>
#include <seqan3/alphabet/nucleotide/dna5.hpp>
#include <seqan3/core/debug_stream.hpp>
#include <seqan3/io/record.hpp>
#include <seqan3/io/sam_file/input.hpp>
#include <seqan3/io/sequence_file/input.hpp>
int main()
{
std::filesystem::path current_path = std::filesystem::current_path();
// read in reference information
seqan3::sequence_file_input reference_file{current_path / "reference.fasta"};
std::vector<std::string> reference_ids{};
std::vector<seqan3::dna5_vector> reference_sequences{};
for (auto && record : reference_file)
{
reference_ids.push_back(std::move(record.id()));
reference_sequences.push_back(std::move(record.sequence()));
}
// filter out alignments
seqan3::sam_file_input mapping_file{current_path / "mapping.sam", reference_ids, reference_sequences};
auto mapq_filter = std::views::filter(
[](auto & record)
{
return record.mapping_quality() >= 30;
});
for (auto & record : mapping_file | mapq_filter)
{
auto alignment = seqan3::alignment_from_cigar(record.cigar_sequence(),
reference_sequences[record.reference_id().value()],
record.reference_position().value(),
record.sequence());
// as loop
size_t sum_reference{};
for (auto const & char_reference : std::get<0>(alignment))
if (char_reference == seqan3::gap{})
++sum_reference;
// or via std::ranges::count
size_t sum_read = std::ranges::count(std::get<1>(alignment), seqan3::gap{});
// The reference_id is ZERO based and an optional. -1 is represented by std::nullopt (= reference not known).
std::optional reference_id = record.reference_id();
seqan3::debug_stream << record.id() << " mapped against "
<< (reference_id ? std::to_string(reference_id.value()) : "unknown reference") << " with "
<< sum_read << " gaps in the read sequence and " << sum_reference
<< " gaps in the reference sequence.\n";
}
}

# Writing alignment files

## Writing records

When writing a SAM file without any further specifications, the default file assumes that all fields are provided. Since those are quite a lot for alignment files, we usually want to write only a subset of the data stored in the SAM format and default the rest.

For this purpose, you can use the seqan3::sam_record to write out a partial record.

#include <seqan3/io/sam_file/all.hpp>
int main()
{
using namespace seqan3::literals;
auto filename = std::filesystem::current_path() / "out.sam";
seqan3::sam_file_output fout{filename};
using types = seqan3::type_list<std::vector<seqan3::dna5>, std::string, std::vector<seqan3::cigar>>;
using fields = seqan3::fields<seqan3::field::seq, seqan3::field::id, seqan3::field::cigar>;
using sam_record_type = seqan3::sam_record<types, fields>;
// write the following to the file
// r001 0 * 0 0 4M2I2M2D * 0 0 ACGTACGT *
sam_record_type record{};
record.id() = "r001";
record.sequence() = "ACGTACGT"_dna5;
record.cigar_sequence() = {{4, 'M'_cigar_operation},
{2, 'I'_cigar_operation},
{2, 'M'_cigar_operation},
{2, 'D'_cigar_operation}};
fout.push_back(record);
}

Note that this only works because in the SAM format all fields are optional. If we provide fewer fields when writing, default values are written.

#### Assignment 3: Writing id and sequence information

Create a small program that writes the following unmapped (see seqan3::sam_flag) read ids and sequences:

read1: ACGATCGACTAGCTACGATCAGCTAGCAG
read2: AGAAAGAGCGAGGCTATTTTAGCGAGTTA

Your ids can be of type std::string and your sequences of type std::vector<seqan3::dna4>.

Your resulting SAM file should look like this:

read1 4 * 0 0 * * 0 0 ACGATCGACTAGCTACGATCAGCTAGCAG *
read2 4 * 0 0 * * 0 0 AGAAAGAGCGAGGCTATTTTAGCGAGTTA *

Solution
#include <filesystem>
#include <seqan3/alphabet/nucleotide/dna4.hpp>
#include <seqan3/io/sam_file/all.hpp>
int main()
{
using namespace seqan3::literals;
auto filename = std::filesystem::current_path() / "my.sam";
std::vector<std::string> ids{"read1", "read2"};
std::vector<std::vector<seqan3::dna4>> seqs{"ACGATCGACTAGCTACGATCAGCTAGCAG"_dna4,
"AGAAAGAGCGAGGCTATTTTAGCGAGTTA"_dna4};
seqan3::sam_file_output fout{filename};
using types = seqan3::type_list<std::string &, std::vector<seqan3::dna4> &, seqan3::sam_flag>;
using fields = seqan3::fields<seqan3::field::id, seqan3::field::seq, seqan3::field::flag>;
using sam_record_type = seqan3::sam_record<types, fields>;
for (size_t i = 0; i < ids.size(); ++i)
{
fout.push_back(sam_record_type{ids[i], seqs[i], seqan3::sam_flag::unmapped});
}
}

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