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Working with biological sequences


Teaching: XX min
Exercises: XX min
  • What is the recommended way to represent biological sequences in Bioconductor?

  • What Bioconductor packages provides methods to efficiently process biological sequences?

  • Explain how biological sequences are represented in the Bioconductor project.

  • Identify Bioconductor packages and methods available to process biological sequences.

Install packages

Before we can proceed into the following sections, we install some Bioconductor packages that we will need. First, we check that the BiocManager package is installed before trying to use it; otherwise we install it. Then we use the BiocManager::install() function to install the necessary packages.

if (!requireNamespace("BiocManager", quietly = TRUE))


The Biostrings package and classes

Why do we need classes for biological sequences?

Biological sequences are arguably some of the simplest biological entities to represent computationally. Examples include nucleic acid sequences (e.g., DNA, RNA) and protein sequences composed of amino acids.

That is because alphabets have been designed and agreed upon to represent individual monomers using character symbols.

For instance, using the alphabet for amino acids, the reference protein sequence for the Actin, alpha skeletal muscle protein sequence is represented as follows.


However, a major limitation of regular character vectors is that they do not check the validity of the sequences that they contain. Practically, it is possible to store meaningless sequences of symbols in character strings, including symbols that are not part of the official alphabet the relevant type of polymer. In those cases, the burden of checking the validity of sequences falls on the programs that process them, or causing those programs to run into errors when they unexpectedly encounter invalid symbols in a sequence.

Instead, S4 classes – demonstrated in the earlier episode The S4 class system – provide a way to label objects as distinct “DNA”, “RNA”, or “protein” varieties of biological sequences. This label is an extremely powerful way to inform programs on the set of character symbols they can expect in the sequence, but also the range of computational operations that can be applied to those sequences. For instance, a function designed to translate nucleic acid sequences into the corresponding amino acid sequence should only be allowed to run on sequences that represent nucleic acids.


Can you tell whether this character string is a valid DNA sequence?



Yes, this is a valid DNA sequence using ambiguity codes defined in the IUPAC notation. In this case, A, T, C, and G represents the four standard types of nucleotides, while the R symbol acts as a regular expression representing either of the two purine nucleotide bases, A and G.

The Biostrings package


In the Bioconductor project, the Biostrings package implements S4 classes to represent biological sequences as S4 objects, e.g. DNAString for sequences of nucleotides in deoxyribonucleic acid polymers, and AAString for sequences of amino acids in protein polymers. Those S4 classes provide memory-efficient containers for character strings, automatic validity-checking functionality for each class of biological molecules, and methods implementing various string matching algorithms and other utilities for fast manipulation and processing of large biological sequences or sets of sequences.

A short presentation of the basic classes defined in the Biostrings package is available in one of the package vignettes, accessible as vignette("Biostrings2Classes"), while more detailed information is provided in the other package vignettes, accessible as browseVignettes("Biostrings").

First steps

To get started, we load the package.


With the package loaded and attached to the session, we have access to the package functions. Those include functions that let us create new objects of the classes defined in the package. For instance, we can create an object that represents a DNA sequence, using the DNAString() constructor function. Without assigning the output to an object, we let the resulting object be printed in the console.

4-letter DNAString object
seq: ATCG

Notably, DNA sequences may only contain the symbols A, T, C, and G, to represent the four DNA nucleotide bases, the symbol N as a placeholder for an unknown or unspecified base, and a restricted set of additional symbols with special meaning defined in the IUPAC Extended Genetic Alphabet. Notice that the constructor function does not let us create objects that contain invalid characters, e.g. Z.

Error in .Call2("new_XString_from_CHARACTER", class(x0), string, start, : key 90 (char 'Z') not in lookup table

Specifically, the IUPAC Extended Genetic Alphabet defines ambiguity codes that represent sets of nucleotides, in a way similar to regular expressions. The IUPAC_CODE_MAP named character vector contains the mapping from the IUPAC nucleotide ambiguity codes to their meaning.

     A      C      G      T      M      R      W      S      Y      K      V      H      D      B      N 
   "A"    "C"    "G"    "T"   "AC"   "AG"   "AT"   "CG"   "CT"   "GT"  "ACG"  "ACT"  "AGT"  "CGT" "ACGT" 

Any of those nucleotide codes are allowed in the sequence of a DNAString object. For instance, the symbol M represents either of the two nucleotides A or C at a given position in a nucleic acid sequence.

5-letter DNAString object
seq: ATCGM

In particular, pattern matching methods implemented in the Biostrings package recognize the meaning of ambiguity codes for each class of biological sequence, allowing them to efficiently match motifs queried by users without the need to design elaborate regular expressions. For instance, the method matchPattern() takes a pattern= and a subject= argument, and returns a Views object that reports and displays any match of the pattern expression at any position in the subject sequence.

Note that the default option fixed = TRUE instructs the method to match the query exactly – i.e., ignore ambiguity codes – which in this case does report any exact match.

dna1 <- DNAString("ATCGCTTTGA")
matchPattern("GM", dna1, fixed = TRUE)
Views on a 10-letter DNAString subject
views: NONE

Instead, to indicate that the pattern includes some ambiguity code, the argument fixed must be set to FALSE.

matchPattern("GM", dna1, fixed = FALSE)
Views on a 10-letter DNAString subject
      start end width
  [1]     4   5     2 [GC]
  [2]     9  10     2 [GA]

In this particular example, two views describes matches of the pattern in the subject sequence. Specifically, the pattern GM first matched the sequence GC spanning positions 4 to 5 in the subject sequence, and then also matched the sequence GA from positions 9 to 10.

Similarly to the method matchPattern(), the method countPattern() can be applied to simply count the number of matches of the pattern in the subject sequence. And again, the option fixed controls whether to respect ambiguity codes, or match them exactly.


How many hits does the following code return? Why?

countPattern("GM", dna2, fixed = FALSE)


The method countPattern() reports 3 hits, because the option fixed = FALSE allow the pattern GM to match GA, GC, and GM sequences, due to the use of the ambiguity code M in the pattern.

Importing biological strings from files

In practice, users rarely type the strings representing biological sequences themselves. Most of the time, biological strings are imported from files, either downloaded from public repositories or generated locally using bioinformatics programs.

For instance, we can load the set of adapter sequences for the TruSeq™ DNA PCR-Free whole-genome sequencing library preparation kit from a file that we downloaded the file during the lesson setup. Since adapter sequences are nucleic acid sequences, we must use the function readDNAStringSet().

truseq_adapters <- readDNAStringSet(filepath = "data/TruSeq3-PE-2.fa")
DNAStringSet object of length 6:
    width seq                                                                                       names               
[1]    34 TACACTCTTTCCCTACACGACGCTCTTCCGATCT                                                        PrefixPE/1
[2]    34 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT                                                        PrefixPE/2
[3]    34 TACACTCTTTCCCTACACGACGCTCTTCCGATCT                                                        PE1
[4]    34 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA                                                        PE1_rc
[5]    34 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT                                                        PE2
[6]    34 AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC                                                        PE2_rc

Going further

The help page of the function readDNAStringSet() – accessible using help(readDNAStringSet) – documents related functions designed to import other types of biological sequences, e.g readRNAStringSet(), readAAStringSet().

Operations on biological strings

Computing the frequency of symbols

The Biostrings package provides several functions to process and manipulate classes of biological strings. For example, we have come across matchPattern() and countPattern() earlier in this episode.

Another example of method that can be applied to biological strings is to compute the frequency of letters in a biological sequence, using the method letterFrequency().

letterFrequency(truseq_adapters, letters = DNA_ALPHABET)
      A  C  G  T M R W S Y K V H D B N - + .
[1,]  6 14  3 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0
[2,]  5  8 10 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0
[3,]  6 14  3 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0
[4,] 11  3 14  6 0 0 0 0 0 0 0 0 0 0 0 0 0 0
[5,]  5  8 10 11 0 0 0 0 0 0 0 0 0 0 0 0 0 0
[6,] 11 10  8  5 0 0 0 0 0 0 0 0 0 0 0 0 0 0

The output is a matrix with one row for each sequence in the DNAStringSet object, and one column for each symbol in the alphabet of deoxyribonucleic acids, provided by the Biostrings package in a built-in object called DNA_ALPHABET.

Amino acid sequences

Similarly to the DNAString and DNAStringSet classes, the classes AAString and AAStringSet allow efficient storage and manipulation of a long amino acid sequence, or a set thereof.

Similarly to built-in objects for the DNA alphabet, the built-in objects AA_ALPHABET, AA_STANDARD and AA_PROTEINOGENIC describe different subsets of the alphabet of valid symbols for amino acid sequences.

For instance, the AA_ALPHABET object describes the set of symbols in the full amino acid alphabet.

 [1] "A" "R" "N" "D" "C" "Q" "E" "G" "H" "I" "L" "K" "M" "F" "P" "S" "T" "W" "Y" "V" "U" "O" "B" "J" "Z" "X" "*" "-" "+"
[30] "."


Use base R code to identify the two symbols present in the AA_PROTEINOGENIC alphabet object that are absent from the AA_STANDARD alphabet object. What do those two symbols represent?


[1] "U" "O"

The symbols U and O represent selenocysteine and pyrrolysine, respectively. Those two amino acids are in some species coded for by codons that are usually interpreted as stop codons. As such, they are not included in the alphabet of “standard” amino acids, and a alphabet of “proteinogenic” amino acids was defined to acknowledge the special biology of those amino acids. Either of those alphabets may be used to determine the validity of an amino acid sequence, depending on its biological nature.

Translating nucleotide sequences

One of the key motivations for the use of S4 classes and the object-oriented programming (OOP) model relies on the infrastructure of S4 generics and methods. As described in the earlier episode The S4 class system, generics provide a mechanism for defining and applying distinct implementations of the same generic function name, according to the nature of the input object(s) provided to the function call.

For instance, the Biostrings package provide multiple implementations of a generic called translate(), for translating DNA or RNA sequences into amino acid sequences. The set of input objects supported by the generic translate() can be listed using the function showMethods(), from the CRAN package methods.

Function: translate (package Biostrings)

In the output above, we see that that the generic function translate() includes methods capable of handling objects representing DNA and RNA sequences in the DNAString and RNAString classes, respectively; but also lists of DNA and RNA sequences in objects of class DNAStringSet and RNAStringSet, as well as other classes capable of storing DNA and RNA sequences.

To demonstrate the use of the translate() method, we first load a set of open reading frame (ORFs) identified by the NIH Open Reading Frame Finder for the Homo sapiens actin beta (ACTB) mRNA (RefSeq: NM_001101), using the standard genetic code, a minimal ORF length of 75 nucleotide, and starting with the “ATG” start codon only.

actb_orf_nih <- readDNAStringSet("data/actb_orfs.fasta")
Error in .Call2("new_input_filexp", filepath, PACKAGE = "XVector"): cannot open file 'data/actb_orfs.fasta'
Error in eval(expr, envir, enclos): object 'actb_orf_nih' not found

Having imported the nucleotide sequences as a DNAStringSet object, we can apply the translate() method to that object, to produce the amino acid sequence that results from the translation process for each nucleotide sequence.

actb_aa <- translate(actb_orf_nih)
Error in h(simpleError(msg, call)): error in evaluating the argument 'x' in selecting a method for function 'translate': object 'actb_orf_nih' not found
Error in eval(expr, envir, enclos): object 'actb_aa' not found

In the example above, all amino acid sequences visible start with the typical methionin amino acid encoded by the “ATG” start codon. We also see that all but one of the amini acid sequences visible end with the * symbol, which indicates that the translation process ended on a stop codon. In contrast, the first open reading frame above reached the end of the nucleotide sequence without encoutering a stop codon.

Conveniently, the number of amino acids in each sequence is stated under the header width.


Extract the length of each amino acid sequence above as an integer vector. What is the length of the longest amino acid sequence translated from any of those open reading frame?

Compare your result with the sequence information on the UniPro page for ACTB (


# or

The longest translated sequence contains 376 amino acids.

The Uniprot page reports a sequence of 375 amino acids. However, the UniProt amino acid sequence does not comprise any symbol to represent the stop codon. That difference aside, the UniPro amino acid sequence is identical to the sequence that we was produced by the translate() method.

Key Points

  • The Biostrings package defines classes to represent sequences of nucleotides and amino acids.

  • The Biostrings package also defines methods to efficiently process biological sequences.

  • The BSgenome package provides genome sequences for a range of model organisms immediately available as Bioconductor objects.