# Bio.Seq: Biological Sequences

The Bio.Seq module provides several data types for handling biological symbols and sequences.

## Biological symbols

The Bio.Seq module provides three biological symbol (character) types:

Type Meaning
DNANucleotide DNA nucleotide
RNANucleotide RNA nucleotide
AminoAcid Amino acid

These symbols can be elements of sequences like characters can be elements of strings. See sections beginning from Overview of sequences types section for details.

### DNA and RNA nucleotides

Set of nucleotide symbols in Bio.jl covers IUPAC nucleotide base plus a gap symbol:

Symbol Constant Meaning
'A' DNA_A / RNA_A A; Adenine
'C' DNA_C / RNA_C C; Cytosine
'G' DNA_G / RNA_G G; Guanine
'T' DNA_T T; Thymine (DNA only)
'U' RNA_U U; Uracil (RNA only)
'M' DNA_M / RNA_M A or C
'R' DNA_R / RNA_R A or G
'W' DNA_W / RNA_W A or T/U
'S' DNA_S / RNA_S C or G
'Y' DNA_Y / RNA_Y C or T/U
'K' DNA_K / RNA_K G or T/U
'V' DNA_V / RNA_V A or C or G; not T/U
'H' DNA_H / RNA_H A or C or T; not G
'D' DNA_D / RNA_D A or G or T/U; not C
'B' DNA_B / RNA_B C or G or T/U; not A
'N' DNA_N / RNA_N A or C or G or T/U
'-' DNA_Gap / RNA_Gap Gap (none of the above)

http://www.insdc.org/documents/feature_table.html#7.4.1

Symbols are accessible as constants with DNA_ or RNA_ prefix:

julia> DNA_A
DNA_A

julia> DNA_T
DNA_T

julia> RNA_U
RNA_U

julia> DNA_Gap
DNA_Gap

julia> typeof(DNA_A)
Bio.Seq.DNANucleotide

julia> typeof(RNA_A)
Bio.Seq.RNANucleotide


Symbols can be constructed by converting regular characters:

julia> convert(DNANucleotide, 'C')
DNA_C

julia> convert(DNANucleotide, 'C') === DNA_C
true


Every nucleotide is encoded using the lower 4 bits of a byte. An unambiguous nucleotide has only one set bit and the other bits are unset. The table below summarizes all unambiguous nucleotides and their corresponding bits. An ambiguous nucleotide is the bitwise OR of unambiguous nucleotides that the ambiguous nucleotide can take. For example, DNA_R (meaning the nucleotide is either DNA_A or DNA_G) is encoded as 0101 because 0101 is the bitwise OR of 0001 (DNA_A) and 0100 (DNA_G). The gap symbol is always 0000.

Nucleotide Bits
DNA_A, RNA_A 0001
DNA_C, RNA_C 0010
DNA_G, RNA_G 0100
DNA_T, RNA_U 1000

The next examples demonstrate bit operations of DNA:

julia> bits(reinterpret(UInt8, DNA_A))
"00000001"

julia> bits(reinterpret(UInt8, DNA_G))
"00000100"

julia> bits(reinterpret(UInt8, DNA_R))
"00000101"

julia> bits(reinterpret(UInt8, DNA_B))
"00001110"

julia> ~DNA_A
DNA_B

julia> DNA_A | DNA_G
DNA_R

julia> DNA_R & DNA_B
DNA_G


### Amino acids

Set of amino acid symbols also covers IUPAC amino acid symbols plus a gap symbol:

Symbol Constant Meaning
'A' AA_A Alanine
'R' AA_R Arginine
'N' AA_N Asparagine
'D' AA_D Aspartic acid (Aspartate)
'C' AA_C Cysteine
'Q' AA_Q Glutamine
'E' AA_E Glutamic acid (Glutamate)
'G' AA_G Glycine
'H' AA_H Histidine
'I' AA_I Isoleucine
'L' AA_L Leucine
'K' AA_K Lysine
'M' AA_M Methionine
'F' AA_F Phenylalanine
'P' AA_P Proline
'S' AA_S Serine
'T' AA_T Threonine
'W' AA_W Tryptophan
'Y' AA_Y Tyrosine
'V' AA_V Valine
'O' AA_O Pyrrolysine
'U' AA_U Selenocysteine
'B' AA_B Aspartic acid or Asparagine
'J' AA_J Leucine or Isoleucine
'Z' AA_Z Glutamine or Glutamic acid
'X' AA_X Any amino acid
'*' AA_Term Termination codon
'-' AA_Gap Gap (none of the above)

http://www.insdc.org/documents/feature_table.html#7.4.3

Symbols are accessible as constants with AA_ prefix:

julia> AA_A
AA_A

julia> AA_Q
AA_Q

julia> AA_Term
AA_Term

julia> typeof(AA_A)
Bio.Seq.AminoAcid


Symbols can be constructed by converting regular characters:

julia> convert(AminoAcid, 'A')
AA_A

julia> convert(AminoAcid, 'P') === AA_P
true


### Other functions

# Bio.Seq.alphabetFunction.

alphabet(typ)


Return an iterator of symbols of typ.

typ is one of DNANucleotide, RNANucleotide, or AminoAcid.

# Bio.Seq.gapFunction.

gap(typ)


Return the gap symbol of typ.

typ is one of DNANucleotide, RNANucleotide, AminoAcid, or Char.

# Bio.Seq.iscompatibleFunction.

iscompatible(x, y)


Return true if and only if x and y are compatible with each other (i.e. x and y can be the same symbol).

x and y must be the same type (DNANucleotide, RNANucleotide or AminoAcid).

Examples

julia> iscompatible(DNA_A, DNA_A)
true

julia> iscompatible(DNA_C, DNA_N)  # DNA_N can be DNA_C
true

julia> iscompatible(DNA_C, DNA_R)  # DNA_R (A or G) cannot be DNA_C
false

julia> iscompatible(AA_A, AA_X)    # AA_X can be AA_A
true


# Bio.Seq.isambiguousFunction.

isambiguous(nt::Nucleotide)


Test if nt is ambiguous nucleotide.

## Overview of sequences types

The Bio.Seq module provides representations and tools for manipulating nucleotide and amino acid sequences. Sequences in Bio.jl are more strictly typed than in many other libraries; elements in a sequence are typed as biological symbol instead of character or byte. They are special purpose types rather than simply strings and hence offer additional functionality that naive string types don't have. Though this strictness sacrifices some convenience, it also means you can always rely on a DNA sequence type to store DNA and nothing but DNA, without having to check, or deal with lowercase versus uppercase and so on. Strict separation of sequence types also means we are free to choose the most efficient representation. DNA and RNA sequences are encoded using four bits per base by default making them memory efficient, and also allowing us to speed up many common operations like nucleotide composition, reverse complement, and k-mer enumeration.

The Bio.Seq provides three different sequence types: BioSequence, Kmer and ReferenceSequence. Each of these types is a subtype of an abstract type called Sequence and supports various string-like operations such as random access and iteration. Different sequence types have different features. In most situations, BioSequence type will do and is used as the default representation. But sometimes other types are much more preferable in terms of memory efficiency and computation performance. Here is the summary table of these three types:

Type Description Element type Mutability Allocation
BioSequence{A<:Alphabet} general-purpose biological sequences DNA, RNA, Amino acids mutable heap
Kmer{T<:Nucleotide,k} specialized for short nucleotide sequences DNA, RNA immutable stack / register
ReferenceSequence specialized for long reference genomes DNA immutable heap

Details of these different representations are explained in the following sections:

## General-purpose sequences

BioSequence{A} is a generic sequence type parameterized by an alphabet type A that defines the domain (or set) of biological symbols, and each alphabet has an associated symbol type. For example, AminoAcidAlphabet is associated with AminoAcid and hence an object of the BioSequence{AminoAcidAlphabet} type represents a sequence of amino acids. Symbols from multiple alphabets can't be intermixed in one sequence type.

The following table summarizes common sequence types that are defined in the Bio.Seq module:

Type Symbol type Type alias
BioSequence{DNAAlphabet{4}} DNANucleotide DNASequence
BioSequence{RNAAlphabet{4}} RNANucleotide RNASequence
BioSequence{AminoAcidAlphabet} AminoAcid AminoAcidSequence
BioSequence{CharAlphabet} Char CharSequence

Parameterized definition of the BioSequence{A} type is for the purpose of unifying the data structure and operations of any symbol type. In most cases, users don't have to care about it and can use type aliases listed above. However, the alphabet type fixes the internal memory encoding and plays an important role when optimizing performance of a program (see Compact representation section for low-memory encodings). It also enables a user to define their own alphabet only by defining few numbers of methods. This is described in Defining a new alphabet section.

### Constructing sequences

Sequence types corresponding to these alphabets can be constructed a number of different ways. Most immediately, sequence literals can be constructed using the string macros dna, rna, aa, and char:

# String decorators are provided for common sequence types
julia> dna"TACGTANNATC"
11nt DNA Sequence:
TACGTANNATC

julia> rna"AUUUGNCCANU"
11nt RNA Sequence:
AUUUGNCCANU

julia> aa"ARNDCQEGHILKMFPSTWYVX"
21aa Amino Acid Sequence:
ARNDCQEGHILKMFPSTWYVX

julia> char"αβγδϵ"
5char Char Sequence:
αβγδϵ


However it should be noted that by default these sequence literals allocate the BioSequence object before the code containing the sequence literal is run. This means there may be occasions where your program does not behave as you first expect, even though it is the intended behaviour. For example consider the following code:

julia> function foo()

s = dna"CTT"

push!(s, DNA_A)

end
foo (generic function with 1 method)


You might expect that every time you call foo, that a DNA sequence CTTA would be returned. You might expect that this is because every time foo is called, a new DNA sequence variable CTT is created, and and A nucleotide is pushed to it, and the result, CTTA is returned. In other words you might expect the following output:

julia> foo()
4nt DNA Sequence:
CTTA

julia> foo()
4nt DNA Sequence:
CTTA

julia> foo()
4nt DNA Sequence:
CTTA


However, this is not what happens, instead the following happens:

julia> foo()
4nt DNA Sequence:
CTTA

julia> foo()
5nt DNA Sequence:
CTTAA

julia> foo()
6nt DNA Sequence:
CTTAAA


The reason for this is because the sequence literal is allocated only once before the first time the function foo is called and run. Therefore, s in foo is always a reference to that one sequence that was allocated. So one sequence is created before foo is called, and then it is pushed to every time foo is called. Thus, that one allocated sequence grows with every call of foo.

If you wanted foo to create a new sequence each time it is called, then you can add a flag to the end of the sequence literal to dictate behaviour: A flag of 's' means 'static': the sequence will be allocated before code is run, as is the default behaviour described above. However providing 'd' flag changes the behaviour: 'd' means 'dynamic': the sequence will be allocated at whilst the code is running, and not before. So to change foo so as it creates a new sequence each time it is called, simply add the 'd' flag to the sequence literal:

julia> function foo()

s = dna"CTT"d     # 'd' flag appended to the string literal.

push!(s, DNA_A)

end
foo (generic function with 1 method)


Now every time foo is called, a new sequence CTT is created, and an A nucleotide is pushed to it:

julia> foo()
4nt DNA Sequence:
CTTA

julia> foo()
4nt DNA Sequence:
CTTA

julia> foo()
4nt DNA Sequence:
CTTA


So the take come message of sequence literals is: Be careful when you are using sequence literals inside of functions, and inside the bodies of things like for loops. And if you use them and are unsure, use the 's' and 'd' flags to ensure the behaviour you get is the behaviour you intend.

Sequences can also be constructed from strings or arrays of nucleotide or amino acid symbols using constructors or the convert function:

julia> DNASequence("TTANC")
5nt DNA Sequence:
TTANC

julia> DNASequence([DNA_T, DNA_T, DNA_A, DNA_N, DNA_C])
5nt DNA Sequence:
TTANC

julia> convert(DNASequence, [DNA_T, DNA_T, DNA_A, DNA_N, DNA_C])
5nt DNA Sequence:
TTANC


Using convert, these operations are reversible: sequences can be converted to strings or arrays:

julia> convert(String, dna"TTANGTA")
"TTANGTA"

julia> convert(Vector{DNANucleotide}, dna"TTANGTA")
7-element Array{Bio.Seq.DNANucleotide,1}:
DNA_T
DNA_T
DNA_A
DNA_N
DNA_G
DNA_T
DNA_A


Sequences can also be concatenated into longer sequences:

julia> DNASequence(dna"ACGT", dna"NNNN", dna"TGCA")
12nt DNA Sequence:
ACGTNNNNTGCA

julia> dna"ACGT" * dna"TGCA"
8nt DNA Sequence:
ACGTTGCA

julia> repeat(dna"TA", 10)
20nt DNA Sequence:
TATATATATATATATATATA

julia> dna"TA" ^ 10
20nt DNA Sequence:
TATATATATATATATATATA


Despite being separate types, DNASequence and RNASequence can freely be converted between efficiently without copying the underlying data:

julia> dna = dna"TTANGTAGACCG"
12nt DNA Sequence:
TTANGTAGACCG

julia> rna = convert(RNASequence, dna)
12nt RNA Sequence:
UUANGUAGACCG

julia> dna.data === rna.data  # underlying data are same
true


A random sequence can be obtained by the randdnaseq, randrnaseq and randaaseq functions, which generate DNASequence, RNASequence and AminoAcidSequence, respectively. Generated sequences are composed of the standard symbols without ambiguity and gap. For example, randdnaseq(6) may generate dna"TCATAG" but never generates dna"TNANAG" or dna"T-ATAG".

A translatable RNASequence can also be converted to an AminoAcidSequence using the translate function described below.

### Indexing and modifying

Sequences for the most part behave like other vector or string types. They can be indexed using integers or ranges:

julia> seq = dna"ACGTTTANAGTNNAGTACC"
19nt DNA Sequence:
ACGTTTANAGTNNAGTACC

julia> seq[5]
DNA_T

julia> seq[6:end]
14nt DNA Sequence:
TANAGTNNAGTACC


Indexing by range creates a subsequence of the original sequence. Unlike Vector in the standard library, creating a subsequences is copy-free: a subsequence is just a reference to the original sequence with its range. You may think that this is unsafe because modifying subsequences propagates to the original sequence, but this doesn't happen actually:

julia> seq = dna"AAAA"    # create a sequence
4nt DNA Sequence:
AAAA

julia> subseq = seq[1:2]  # create a subsequence from seq
2nt DNA Sequence:
AA

julia> subseq[2] = DNA_T  # modify the second element of it
DNA_T

julia> subseq             # the subsequence is modified
2nt DNA Sequence:
AT

julia> seq                # but the original sequence is not
4nt DNA Sequence:
AAAA


This is because modifying a sequence checks whether its underlying data are shared with other sequences under the hood. If and only if the data are shared, the subsequence creates a copy of itself. Any modifying operation does this check. This is called copy-on-write strategy and users don't need to care about it because it is transparent from outward.

The following modifying operations are currently supported:

setindex!(seq, item, index)
push!(seq, item)
pop!(seq)
shift!(seq)
unshift!(seq, item)
insert!(seq, index, item)
deleteat!(seq, index)
append!(seq, other_seq)
copy!(dst_seq, dest_offset, src_seq, src_offset, len)
reverse!(seq)


And these two are functions for nucleotide sequences:

complement!(seq)
reverse_complement!(seq)

julia> seq = dna"ACG"
3nt DNA Sequence:
ACG

julia> push!(seq, DNA_T)
4nt DNA Sequence:
ACGT

julia> append!(seq, dna"AT")
6nt DNA Sequence:
ACGTAT

julia> reverse!(seq)
6nt DNA Sequence:
TATGCA

julia> complement!(seq)
6nt DNA Sequence:
ATACGT

julia> reverse_complement!(seq)
6nt DNA Sequence:
ACGTAT


Sequences also work as iterators over symbols:

julia> n = 0
0

julia> for nt in dna"ATNGNNT"

if nt == DNA_N

n += 1

end

end

julia> n
3


### Other operations on sequences

A number of common sequence operations are provided in the Bio.Seq module:

# Bio.Seq.complementFunction.

complement(nt::Nucleotide)


Return the complementary nucleotide of nt.

complement(seq)


Make a complement sequence of seq.

complement(kmer::Kmer)


Return the complement of kmer.

# Bio.Seq.reverse_complementFunction.

reverse_complement(seq)


Make a reversed complement sequence of seq.

Ambiguous nucleotides are left as-is.

reverse_complement(kmer::Kmer)


Return the reverse complement of kmer

# Bio.Seq.mismatchesFunction.

mismatches(seq1::BioSequence, seq2::BioSequence[, compatible=false])


Return the number of mismatches between seq1 and seq2.

If seq1 and seq2 are of differing lengths, only the first min(length(seq1), length(seq2)) nucleotides are compared. When compatible is true, sequence symbols are comapred using iscompatible; otherwise using ==.

mismatches(a::Kmer, b::Kmer)


Return the number of mismatches between a and b.

# Bio.Seq.compositionFunction.

composition(seq | kmer_iter)


Calculate composition of biological symbols in seq or k-mers in kmer_iter.

# Bio.Seq.seqmatrixFunction.

seqmatrix{A<:Alphabet}(vseq::Vector{BioSequence{A}}, major::Symbol)


Construct a matrix of nucleotides or amino acids from a vector of BioSequences.

If parameter major is set to :site, the matrix is created such that one nucleotide from each sequence is placed in each column i.e. the matrix is laid out in site-major order. This means that iteration over one position of many sequences is efficient, as julia arrays are laid out in column major order.

If the parameter major is set to :seq, the matrix is created such that each sequence is placed in one column i.e. the matrix is laid out in sequence-major order. This means that iteration across each sequence in turn is efficient, as julia arrays are laid out in column major order.

Examples

julia> seqs = [dna"AAA", dna"TTT", dna"CCC", dna"GGG"]
4-element Array{Bio.Seq.BioSequence{Bio.Seq.DNAAlphabet{4}},1}:
3nt DNA Sequence:
AAA
3nt DNA Sequence:
TTT
3nt DNA Sequence:
CCC
3nt DNA Sequence:
GGG

julia> seqmatrix(seqs, :site)
4x3 Array{Bio.Seq.DNANucleotide,2}:
DNA_A  DNA_A  DNA_A
DNA_T  DNA_T  DNA_T
DNA_C  DNA_C  DNA_C
DNA_G  DNA_G  DNA_G

julia> seqmatrix(seqs, :seq)
3x4 Array{Bio.Seq.DNANucleotide,2}:
DNA_A  DNA_T  DNA_C  DNA_G
DNA_A  DNA_T  DNA_C  DNA_G
DNA_A  DNA_T  DNA_C  DNA_G

seqmatrix{A<:Alphabet,T}(::Type{T}, vseq::Vector{BioSequence{A}}, major::Symbol)


Construct a matrix of T from a vector of BioSequences.

If parameter major is set to :site, the matrix is created such that one nucleotide from each sequence is placed in each column i.e. the matrix is laid out in site-major order. This means that iteration over one position of many sequences is efficient, as julia arrays are laid out in column major order.

If the parameter major is set to :seq, the matrix is created such that each sequence is placed in one column i.e. the matrix is laid out in sequence-major order. This means that iteration across each sequence in turn is efficient, as julia arrays are laid out in column major order.

Examples

julia> seqs = [dna"AAA", dna"TTT", dna"CCC", dna"GGG"]
4-element Array{Bio.Seq.BioSequence{Bio.Seq.DNAAlphabet{4}},1}:
3nt DNA Sequence:
AAA
3nt DNA Sequence:
TTT
3nt DNA Sequence:
CCC
3nt DNA Sequence:
GGG

julia> seqmatrix(seqs, :site, UInt8)
4×3 Array{UInt8,2}:
0x01  0x01  0x01
0x08  0x08  0x08
0x02  0x02  0x02
0x04  0x04  0x04

julia> seqmatrix(seqs, :seq, UInt8)
3×4 Array{UInt8,2}:
0x01  0x08  0x02  0x04
0x01  0x08  0x02  0x04
0x01  0x08  0x02  0x04


#### Translation

The translate funtion translates a sequence of codons in a RNA sequence to a amino acid sequence besed on a genetic code mapping. The Bio.Seq module contains all NCBI defined genetic codes and they are registered in ncbi_trans_table.

# Bio.Seq.translateFunction.

translate(rna_seq, code=standard_genetic_code, allow_ambiguous_codons=true)


Translate an RNASequence to an AminoAcidSequence.

Translation uses genetic code code to map codons to amino acids. See ncbi_trans_table for available genetic codes. If codons in the given RNA sequence cannot determine a unique amino acid, they will be translated to AA_X if allow_ambiguous_codons is true and otherwise result in an error.

# Bio.Seq.ncbi_trans_tableConstant.

Genetic code list of NCBI.

The standard genetic code is ncbi_trans_table[1] and others can be shown by show(ncbi_trans_table). For more details, consult the next link: http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=cgencodes.

julia> ncbi_trans_table
Translation Tables:
1. The Standard Code (standard_genetic_code)
2. The Vertebrate Mitochondrial Code (vertebrate_mitochondrial_genetic_code)
3. The Yeast Mitochondrial Code (yeast_mitochondrial_genetic_code)
4. The Mold, Protozoan, and Coelenterate Mitochondrial Code and the Mycoplasma/Spiroplasma Code (mold_mitochondrial_genetic_code)
5. The Invertebrate Mitochondrial Code (invertebrate_mitochondrial_genetic_code)
6. The Ciliate, Dasycladacean and Hexamita Nuclear Code (ciliate_nuclear_genetic_code)
9. The Echinoderm and Flatworm Mitochondrial Code (echinoderm_mitochondrial_genetic_code)
10. The Euplotid Nuclear Code (euplotid_nuclear_genetic_code)
11. The Bacterial, Archaeal and Plant Plastid Code (bacterial_plastid_genetic_code)
12. The Alternative Yeast Nuclear Code (alternative_yeast_nuclear_genetic_code)
13. The Ascidian Mitochondrial Code (ascidian_mitochondrial_genetic_code)
14. The Alternative Flatworm Mitochondrial Code (alternative_flatworm_mitochondrial_genetic_code)
16. Chlorophycean Mitochondrial Code (chlorophycean_mitochondrial_genetic_code)
21. Trematode Mitochondrial Code (trematode_mitochondrial_genetic_code)
22. Scenedesmus obliquus Mitochondrial Code (scenedesmus_obliquus_mitochondrial_genetic_code)
23. Thraustochytrium Mitochondrial Code (thraustochytrium_mitochondrial_genetic_code)
24. Pterobranchia Mitochondrial Code (pterobrachia_mitochondrial_genetic_code)
25. Candidate Division SR1 and Gracilibacteria Code (candidate_division_sr1_genetic_code)


http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/index.cgi?chapter=cgencodes

### Compact representation

As we saw above, DNA and RNA sequences can store any ambiguous nucleotides like 'N'. If you are sure that nucleotide sequences store unambiguous nucleotides only, you can save the memory space of sequences. DNAAlphabet{2} is an alphabet that uses two bits per base and limits to only unambiguous nucleotide symbols (ACGT in DNA and ACGU in RNA). To create a sequence of this alphabet, you need to explicitly pass DNAAlphabet{2} to BioSequence as its parametric type:

julia> seq = BioSequence{DNAAlphabet{2}}("ACGT")
4nt DNA Sequence:
ACGT


Recall that DNASequence is a type alias of BioSequence{DNAAlphabet{4}}, which uses four bits per base. That is, BioSequence{DNAAlphabet{2}} saves half of memory footprint compared to BioSequence{DNAAlphabet{4}}. If you need to handle reference genomes that are composed of five nucleotides, ACGTN, consider to use the ReferenceSequence type described in the Reference sequences section.

### Defining a new alphabet

The alphabet type parameter A of BioSequence{A} enables a user to extend functionality of BioSequence with mimimum effort. As an example, definition of a new alphabet type representing a sequence of boolean values is shown below:

using Bio.Seq

immutable BoolAlphabet <: Alphabet end

Seq.bitsof(::Type{BoolAlphabet}) = 1
Seq.eltype(::Type{BoolAlphabet}) = Bool
Seq.alphabet(::Type{BoolAlphabet}) = false:true

function Seq.encode(::Type{BoolAlphabet}, x::Bool)
return UInt64(ifelse(x, 0x01, 0x00))
end

function Seq.decode(::Type{BoolAlphabet}, x::UInt64)
if x > 0x01
throw(Seq.DecodeError(BoolAlphabet, x))
end
return ifelse(x == 0x00, false, true)
end


## Nucleotide k-mers

A common strategy to simplify the analysis of sequence data is to operate or short k-mers, for size fixed size k. These can be packed into machine integers allowing extremely efficient code. The Bio.Seq module has built in support for representing short sequences in 64-bit integers. Besides being fixed length, Kmer types, unlike other sequence types cannot contain ambiguous symbols like 'N'.

The Kmer{T,k} type parameterized on symbol type (T, either DNANucleotide, or RNANucleotide) and size k. For ease of writing code, two type aliases for each nucleotide type are defined and named as DNAKmer{k} and RNAKmer{k}:

julia> DNAKmer("ACGT")  # create a DNA 4-mer from a string
DNA 4-mer:
ACGT

julia> RNAKmer("ACGU")  # create an RNA 4-mer from a string
RNA 4-mer:
ACGU

julia> typeof(DNAKmer("ACGT"))
Bio.Seq.Kmer{Bio.Seq.DNANucleotide,4}


# Bio.Seq.eachFunction.

each(::Type{Kmer{T,k}}, seq::Sequence[, step=1])


Initialize an iterator over all k-mers in a sequence seq skipping ambiguous nucleotides without changing the reading frame.

Arguments

• Kmer{T,k}: k-mer type to enumerate.
• seq: a nucleotide sequence.
• step=1: the number of positions between iterated k-mers

Examples

# iterate over DNA codons
for (pos, codon) in each(DNAKmer{3}, dna"ATCCTANAGNTACT", 3)
@show pos, codon
end


# Bio.Seq.canonicalFunction.

canonical(kmer::Kmer)


Return the canonical k-mer of x.

A canonical k-mer is the numerical lesser of a k-mer and its reverse complement. This is useful in hashing/counting k-mers in data that is not strand specific, and thus observing k-mer is equivalent to observing its reverse complement.

# Bio.Seq.neighborsFunction.

neighbors(kmer::Kmer)


Return an iterator through k-mers neighboring kmer on a de Bruijn graph.

## Reference sequences

DNASequence (alias of BioSequence{DNAAlphabet{4}}) is a flexible data sturcture but always consumes 4 bits per base, which will waste a large part of the memory space when storing reference genome sequences. In such a case, ReferenceSequence is helpful because it compresses positions of 'N' symbols so that long DNA sequences are stored with almost 2 bits per base. An important limitation is that the ReferenceSequence type is immutable due to the compression. Other sequence-like operations are supported:

julia> seq = ReferenceSequence(dna"NNCGTATTTTCN")
12nt Reference Sequence:
NNCGTATTTTCN

julia> seq[1]
DNA_N

julia> seq[5]
DNA_T

julia> seq[2:6]
5nt Reference Sequence:
NCGTA

julia> ReferenceSequence(dna"ATGM")  # DNA_M is not accepted
ERROR: ArgumentError: invalid symbol M ∉ {A,C,G,T,N} at 4
in convert at /Users/kenta/.julia/v0.4/Bio/src/seq/refseq.jl:58
in call at essentials.jl:56


When reading reference sequences from a FASTA file, the following snippet will avoid allocating temporary sequences and conversion:

for record in open(FASTAReader{ReferenceSequence}, "hg38.fa")
# do something
end


Three kinds of on-line search functions are provided:

1. Exact search
2. Approximate search
3. Regular expression search

These are all specialized for biological sequences and ambiguities of symbols are considered.

Exact search functions search for an occurrence of the query symbol or sequence. Four functions, search, searchindex, rsearch, and rsearchindex are available:

julia> seq = dna"ACAGCGTAGCT";

julia> search(seq, DNA_G)  # search a query symbol
4:4

julia> query = dna"AGC";

julia> search(seq, query)  # search a query sequence
3:5

julia> searchindex(seq, query)
3

julia> rsearch(seq, query)  # similar to search but in the reverse direction
8:10

julia> rsearchindex(seq, query)  # similar to searchindex but in the reverse direction
8


These search functions take ambiguous symbols into account. That is, if two symbols are compatible (e.g. DNA_A and DNA_N), they match when searching an occurrence. In the following example, 'N' is a wild card that matches any symbols:

julia> search(dna"ACNT", DNA_N)  # 'A' matches 'N'
1:1

julia> search(dna"ACNT", dna"CGT")  # 'N' matches 'G'
2:4

julia> search(dna"ACGT", dna"CNT")  # 'G' matches 'N'
2:4


The exact sequence search needs preprocessing phase of query sequence before searching phase. This would be enough fast for most search applications. But when searching a query sequence to large amounts of target sequences, caching the result of preprocessing may save time. The ExactSearchQuery creates such a preprocessed query object and is applicable to the search functions:

julia> query = ExactSearchQuery(dna"ATT");

julia> search(dna"ATTTATT", query)
1:3

julia> rsearch(dna"ATTTATT", query)
5:7


The approximate search is similar to the exact search but allows a specific number of errors. That is, it tries to find a subsequence of the target sequence within a specific Levenshtein distance of the query sequence:

julia> seq = dna"ACAGCGTAGCT";

julia> approxsearch(seq, dna"AGGG", 0)  # nothing matches with no errors
0:-1

julia> approxsearch(seq, dna"AGGG", 1)  # seq[3:5] matches with one error
3:6

julia> approxsearch(seq, dna"AGGG", 2)  # seq[1:4] matches with two errors
1:4


Like the exact search functions, four kinds of functions (approxsearch, approxsearchindex, approxrsearch, and approxrsearchindex) are available:

julia> seq = dna"ACAGCGTAGCT"; pat = dna"AGGG";

julia> approxsearch(seq, pat, 2)        # return the range (forward)
1:4

julia> approxsearchindex(seq, pat, 2)   # return the starting index (forward)
1

julia> approxrsearch(seq, pat, 2)       # return the range (backward)
8:11

julia> approxrsearchindex(seq, pat, 2)  # return the starting index (backward)
8


Preprocessing can be cached in an ApproximateSearchQuery object:

julia> query = ApproximateSearchQuery(dna"AGGG");

julia> approxsearch(dna"AAGAGG", query, 1)
2:5

julia> approxsearch(dna"ACTACGT", query, 2)
4:6


Query patterns can be described in regular expressions. The syntax supports a subset of Perl and PROSITE's notation.

The Perl-like syntax starts with biore (biological regular expression) and ends with a symbol option: "dna", "rna" or "aa". For example, biore"A+"dna is a regular expression for DNA sequences and biore"A+"aa is for amino acid sequences. The symbol options can be abbreviated to its first character: "d", "r" or "a", respectively.

Here are examples of using the regular expression for BioSequences:

julia> match(biore"A+C*"dna, dna"AAAACC")
Nullable(RegexMatch("AAAACC"))

julia> match(biore"A+C*"d, dna"AAAACC")
Nullable(RegexMatch("AAAACC"))

julia> ismatch(biore"A+C*"dna, dna"AAC")
true

julia> ismatch(biore"A+C*"dna, dna"C")
false


match always returns a Nullable object and it should be null if no match is found.

The table below summarizes available syntax elements.

Syntax Description Example
\| alternation "A\|T" matches "A" and "T"
* zero or more times repeat "TA*" matches "T", "TA" and "TAA"
+ one or more times repeat "TA+" matches "TA" and "TAA"
? zero or one time "TA?" matches "T" and "TA"
{n,} n or more times repeat "A{3,}" matches "AAA" and "AAAA"
{n,m} n-m times repeat "A{3,5}" matches "AAA", "AAAA" and "AAAAA"
^ the start of the sequence "^TAN*" matches "TATGT"
$ the end of the sequence "N*TA$" matches "GCTA"
(...) pattern grouping "(TA)+" matches "TA" and "TATA"
[...] one of symbols "[ACG]+" matches "AGGC"

eachmatch, matchall, and search are also defined like usual strings:

julia> matchall(biore"TATA*?"d, dna"TATTATAATTA")  # overlap (default)
4-element Array{Bio.Seq.BioSequence{Bio.Seq.DNAAlphabet{4}},1}:
3nt DNA Sequence:
TAT
3nt DNA Sequence:
TAT
4nt DNA Sequence:
TATA
5nt DNA Sequence:
TATAA

julia> matchall(biore"TATA*"d, dna"TATTATAATTA", false)  # no overlap
2-element Array{Bio.Seq.BioSequence{Bio.Seq.DNAAlphabet{4}},1}:
3nt DNA Sequence:
TAT
5nt DNA Sequence:
TATAA

julia> search(dna"TATTATAATTA", biore"TATA*"d)
1:3

julia> search(dna"TATTATAATTA", biore"TATA*"d, 2)
4:8


Notewothy differences from strings are:

• Ambiguous characters match any compatible characters (e.g. biore"N"d is equivalent to biore"[ACGT]"d).
• Whitespaces are ignored (e.g. biore"A C G"d is equivalent to biore"ACG"d).

The PROSITE notation is described in ScanProsite - user manual. The syntax supports almost all notations including the extended syntax. The PROSITE notation starts with prosite prefix and no symbol option is needed because it always descirbe patterns of amino acid sequences:

julia> match(prosite"[AC]-x-V-x(4)-{ED}", aa"CPVPQARG")
Nullable(RegexMatch("CPVPQARG"))

julia> match(prosite"[AC]xVx(4){ED}", aa"CPVPQARG")
Nullable(RegexMatch("CPVPQARG"))


## Sequence composition

Sequence composition can be easily calculated using the composition function:

julia> comp = composition(dna"ACGAG")
DNA Nucleotide Composition:
DNA_A   => 2
DNA_C   => 1
DNA_G   => 2
DNA_T   => 0
DNA_M   => 0
DNA_R   => 0
DNA_W   => 0
DNA_S   => 0
DNA_Y   => 0
DNA_K   => 0
DNA_V   => 0
DNA_H   => 0
DNA_D   => 0
DNA_B   => 0
DNA_N   => 0
DNA_Gap => 0

julia> comp[DNA_A]
2

julia> comp[DNA_T]
0


To accumulate composition statistics of multiple sequences, merge! can be used as follows:

# initiaize an empty composition counter
comp = composition(dna"")

# iterate over sequences and accumulate composition statistics into comp
for seq in seqs
merge!(comp, composition(seq))
end

# or functional programming style in one line
foldl((x, y) -> merge(x, composition(y)), composition(dna""), seqs)


composition is also applicable to a k-mer iterator:

julia> comp = composition(each(DNAKmer{4}, dna"ACGT"^100));

julia> comp[DNAKmer("ACGT")]
100

julia> comp[DNAKmer("CGTA")]
99


## Sequence records

The SeqRecord type is used to represent a named sequence, optionally with accompanying metadata. It is defined as: sequence. It is as:

type SeqRecord{S, T}
name::String
seq::S
end


The type of the metadata field depends on the source of the sequence record. For example, if a record is read from a FASTA file, metadata contains the description field. If from a FASTQ file, a quality scores assigned to base calls during sequencing.

The following accessors are defined for the SeqRecord type:

# Bio.Seq.seqnameFunction.

seqname(record)


Return the sequence name of record.

# Bio.Seq.sequenceFunction.

sequence(record)


Return the sequence of record.

# Bio.Seq.metadataFunction.

metadata(record)


Return the metadata of record.

## Sequence demultiplexing

Multiplex sequencing is a technology to sequence mutliple samples at the same time on a high-throughput DNA sequencer. Samples are distinguished by the short prefix of a DNA sequence called DNA barcode. The Bio.Seq offers the Demultiplexer type and the demultiplex function to identify the DNA barcode of a longer DNA sequence allowing small errors.

In the following example, four kinds of DNA sequences of length 4 are used as DNA barcodes. Demultiplexer takes these barcodes as its first argument with a few options:

julia> barcodes = DNASequence["ATGG", "CAGA", "GGAA", "TACG"];

julia> dplxr = Demultiplexer(barcodes, n_max_errors=1, distance=:hamming)
Bio.Seq.Demultiplexer{Bio.Seq.BioSequence{Bio.Seq.DNAAlphabet{4}}}:
distance: hamming
number of barcodes: 4
number of correctable errors: 1


n_max_errors specifies the number of maximum correctable errors in a barcode. The type of correctable errors depends on the distance parameter. When distance = :hamming as shown above only substitutions are correctable. When distance = :levenshtein substitutions, deletions, and insertions are correctable. The user is responsible for keeping enough distances among barcodes; Demultiplexer will throw an exception if two barcodes are within n_max_errors * 2.

The demultiplex function takes a demultiplexer object and a DNA sequence, and returns a tuple of a barcode index and a distance between the original barcode sequence and the prefix sequence:

julia> demultiplex(dplxr, dna"ATGGCGNT")  # 1st barcode with no errors
(1,0)

julia> demultiplex(dplxr, dna"CAGGCGNT")  # 2nd barcode with one error
(2,1)

julia> demultiplex(dplxr, dna"GGAACGNT")  # 3rd barcode with no errors
(3,0)

julia> demultiplex(dplxr, dna"TGACCGNT")  # no matching barcode
(0,-1)


The optional third argument controls the search strategy. demultiplex uses an index to search the closest barcode within n_max_errors in the barcode set and returns it if any by default. If the third argument is true it falls back to a linear search after the index search and returns one of the closest barcodes at random. The next example shows the difference of these two strategies:

julia> demultiplex(dplxr, dna"TGACCGNT", false)  # linear search off (default)
(0,-1)

julia> demultiplex(dplxr, dna"TGACCGNT", true)   # linear search on
(3,2)