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bch441-work-abc-units/BIN-ALI-MSA.R

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# tocID <- "BIN-ALI-MSA.R"
#
# Purpose: A Bioinformatics Course:
# R code accompanying the BIN-ALI-MSA unit.
#
# Version: 1.3
#
# Date: 2017-10 - 2020-09
# Author: Boris Steipe (boris.steipe@utoronto.ca)
#
# Versions:
# 1.3 2020 updates
# 1.2 Change from require() to requireNamespace(),
# use <package>::<function>() idiom throughout
# 1.1 Added fetchMSAmotif()
# 1.0 Fully refactored and rewritten for 2017
# 0.1 First code copied from 2016 material.
#
#
# TODO:
#
#
# == DO NOT SIMPLY source() THIS FILE! =======================================
#
# If there are portions you don't understand, use R's help system, Google for an
# answer, or ask your instructor. Don't continue if you don't understand what's
# going on. That's not how it works ...
#
# ==============================================================================
#TOC> ==========================================================================
#TOC>
#TOC> Section Title Line
#TOC> ------------------------------------------------------------------
#TOC> 1 Preparations 55
#TOC> 2 Aligning full length MBP1 proteins 97
#TOC> 2.1 Preparing Sequences 108
#TOC> 2.2 Compute the MSA 133
#TOC> 3 Analyzing an MSA 154
#TOC> 4 Comparing MSAs 226
#TOC> 4.1 Importing an alignment to msa 235
#TOC> 4.1.1 importing an .aln file 244
#TOC> 4.1.2 Creating an MsaAAMultipleAlignment object 275
#TOC> 4.2 More alignments 326
#TOC> 4.3 Computing comparison metrics 338
#TOC> 5 Profile-Profile alignments 476
#TOC> 6 Sequence Logos 549
#TOC> 6.1 Subsetting an alignment by motif 558
#TOC> 6.2 Plot a Sequence Logo 607
#TOC>
#TOC> ==========================================================================
# = 1 Preparations ========================================================
# You need to reload you protein database, including changes that might
# have been made to the reference files. If you have worked with the
# prerequisite units, you should have a script named "./myScripts/makeProteinDB.R"
# that will create the myDB object with a protein and feature database.
# Ask for advice if not.
source("./myScripts/makeProteinDB.R")
if (! requireNamespace("BiocManager", quietly=TRUE)) {
install.packages("BiocManager")
}
if (! requireNamespace("Biostrings", quietly=TRUE)) {
BiocManager::install("Biostrings")
}
# Package information:
# library(help = Biostrings) # basic information
# browseVignettes("Biostrings") # available vignettes
# data(package = "Biostrings") # available datasets
# Multiple sequence alignment algorithms are provided in
# the Bioconductor msa package.
if (! requireNamespace("msa", quietly=TRUE)) {
BiocManager::install("msa")
}
# Package information:
# library(help=msa) # basic information
# browseVignettes("msa") # available vignettes
# data(package = "msa") # available datasets
# If an installation asks you if you want to update older packages, I recommend
# to always answer "a" for "all" unless you have an important reason not to. But
# if the installer asks you whether you want to compile from source, answer "n"
# for "no" unless you need new functionality in a particular bleeding-edge
# version of a package.
help(package = "msa")
# = 2 Aligning full length MBP1 proteins ==================================
# In the Wiki part of this unit you have
# - aligned full length MBP1 protein sequences at the EBI using T-Coffee
# - saved the resulting alignment in CLUSTAL format
# to the file "MBP1orthologuesTC.aln"
# In this section we will calculate an MSA of the same sequences using
# algorithms in the msa packages, and we will compare the two alignments.
# == 2.1 Preparing Sequences ===============================================
# We have used the biostrings' AAString() function before; for multiple
# alignments we need an AAStringSet(). AAStringSets are produced from vectors
# of sequence.
sel <- grep("MBP1", myDB$protein$name)
MBP1set <- Biostrings::AAStringSet(myDB$protein$sequence[sel])
# To help us make sense of the alignment we need to add the names for
# the sequences. Names for a seqSet object are held in the ranges slot...
MBP1set@ranges@NAMES <- myDB$protein$name[sel]
MBP1set
# You should have eleven sequences in this set, ask for advice if you don't.
# The little step of adding names is actually really very important. That's
# because the aligned sequences are meaningless strings of characters unless we
# can easily identify their biological relationships. Creating MSAs that are
# only identified by e.g. their RefSeq ids is a type of cargo-cult
# bioinformatics that we encounter a lot. The point of the alignment is not to
# create it, but to interpret it!
# == 2.2 Compute the MSA ===================================================
# The alignment itself is very simple. msa has msaMuscle() and msaClustalOmega()
# to produce alignments. (It also has msaClustalW() which is kind of
# embarrassing since that hasn't been the algorithm of first choice for
# decades. Don't use that one for real work.)
# Let's run an alignment with "Muscle"
(msaM <- msa::msaMuscle( MBP1set, order = "aligned"))
# ... or to see the whole thing (cf. ?MsaAAMultipleAlignment ... print method):
msa::print(msaM, show=c("alignment", "complete"), showConsensus=FALSE)
# You see that the alignment object has sequence strings with hyphens as
# indel-characters. The names are printed to the console. And you also see that
# the order has not been preserved, but the most similar sequences are now
# adjacent to each other.
# = 3 Analyzing an MSA ====================================================
# You probabaly realize that computing an MSA is not that hard. It's not
# entirely trivial to collect meaningful sequences via e.g. PSI-BLAST ... but
# then computing the alignment gives you a result quickly. But what does it
# mean? What information does the MSA contain?
# Let's have a first look at conserved vs. diverged regions of the MSA. msa
# provides the function msaConservationScore() which outputs a vector of scores.
# The scores are the sum of pairscores for the column: for example a perfectly
# conserved column of histidines would have the following score in our MSA
# of eleven sequences:
# - one (H, H) pair score is 8 in BLOSUM62;
# - there are (n^2 - n) / 2 pairs that can be formed between amino acids
# in a column from n sequences;
# - therefore the column score is 8 * (11^2 - 11) / 2 == 440
# attach the BLOSUM62 package from the Biostrings package
data(BLOSUM62, package = "Biostrings")
msaMScores <- msa::msaConservationScore(msaM, substitutionMatrix = BLOSUM62)
plot(msaMScores, type = "l", col = "#205C5E", xlab = "Alignment Position")
# That plot shows the well-aligned regions (domains ?) of the sequence, but it
# could use some smoothing. Options for smoothing such plots include calculating
# averages in sliding windows ("moving average"), and lowess() smoothing. Here
# is a quick demo of a moving average smoothing, to illustrate the principle.
wRadius <- 15 # we take the mean of all values around a point +- wRadius
len <- length(msaMScores)
v <- msaMScores
for (i in (1 + wRadius):(len - wRadius)) {
v[i] <- mean(msaMScores[(i - wRadius):(i + wRadius)]) # mean of values in
# window around i
}
points(v, col = "#FFFFFF", type = "l", lwd = 4.5)
points(v, col = "#3DAEB2", type = "l", lwd = 3)
# You can set a threshold and use rle() to define ranges of values that fall
# above and below the threshold, and thus approximate domain boundaries:
thrsh <- 30
(highScoringRanges <- rle(v > thrsh))
(idx <- c(1, cumsum(highScoringRanges$lengths)))
for (i in seq_along(highScoringRanges$lengths)) {
if (highScoringRanges$values[i] == TRUE) { # If this range is above threshold,
rect(idx[i], thrsh, idx[i+1], max(v), # ... draw a rectangle
col = "#205C5E33") # ... with a transparent color.
cat(sprintf("Possible domain from %d to %d\n", idx[i], idx[i+1]))
}
}
# Getting this right requires a bit of fiddling with the window radius and
# threshold (experiment with that a bit), but once we are satisfied, we can use
# the boundaries to print the MSA alignments separately for domains.
# Unfortunately the msa package provides no native way to extract blocks of the
# alignment for further processing, but your .utilities.R script contains a
# function to write alignment objects and sequence sets to .aln formatted
# output. Have a look:
writeALN
# Print out the aligned blocks
for (i in seq_along(highScoringRanges$lengths)) {
if (highScoringRanges$values[i] == TRUE) { # If this range is above threshold,
cat(sprintf("\n\nPossible domain from %d to %d\n", idx[i], idx[i+1]))
writeALN(msaM, range = c(idx[i], idx[i+1]))
}
}
# = 4 Comparing MSAs ======================================================
# Let's compare the results of different alignment algorithms. We computed a
# vector of scores above, and we can compare that for different alignment
# algorithms. This is not trivial, so we'll need to look at that data in
# different ways and explore it. But first, let's get more alignments to compare
# with.
# == 4.1 Importing an alignment to msa =====================================
# We computed a T-Coffee alignment at the EBI. msa has no native import function
# so we need to improvise, and it's a bit of a pain to do - but a good
# illustration of strategies to convert data into any kind of object:
# - read an .aln file
# - adjust the sequence names
# - convert to msaAAMultipleAlignment object
# === 4.1.1 importing an .aln file
# The seqinr package has a function to read CLUSTAL W formatted .aln files ...
if (! requireNamespace("seqinr", quietly=TRUE)) {
install.packages("seqinr")
}
# Package information:
# library(help=seqinr) # basic information
# browseVignettes("seqinr") # available vignettes
# data(package = "seqinr") # available datasets
# read the T-coffee aligned file that you donwloaded from the EBI MSA tools
# (cf. http://steipe.biochemistry.utoronto.ca/abc/index.php/BIN-ALI-MSA)
tmp <- seqinr::read.alignment("msaT.aln", format = "clustal")
# read.alignment() returns a list. $seq is a list of strings, one for each
# complete alignment. However, they are converted to lower case.
x <- toupper(unlist(tmp$seq)) # get the sequences, uppercase
# $nam contains the names.
(names(x) <- tmp$nam)
# Note that the names in the file are refseq IDs, we need to replace the
# RefSeqIDs with the database names:
for (i in seq_along(x)) {
# find the index of the RefSeqID
id <- gsub("\\..*$", "", names(x)[i]) # fetch the name, drop the version
sel <- which(myDB$protein$RefSeqID == id) # get the index
names(x)[i] <- myDB$protein$name[sel] # get the name
}
# === 4.1.2 Creating an MsaAAMultipleAlignment object
# MsaAAMultipleAlignment objects are S4 objects that contain AAStringSet objects
# in their @unmasked slot, and a few additional items. Rather then build the
# object from scratch, we copy an existing object, and overwrite the data in its
# slots with what we need. Our goal is pragmatic, we want an object that msa's
# functions will accept as input.
# First: convert our named char vector into an AAstringSet
x <- Biostrings::AAStringSet(x)
# Then: create a new MsaAAMultipleAlignment S4 object. The msa package has
# defined what such an object should look like, with the SetClass() function. To
# create a new object, simply use the new() function, define the class that the
# object would have, and fill the slots with something that has the right type.
# How do we know the right type and the slot names? Easy! We just use
# str(<object>) to get the information.
str(msaM)
# There is a catch however in the way R makes such operations specific to
# the packages they need them: the function that creates the class is
# defined as a "generic", and when it is called, R looks in the package
# namespace for a more specific function with precise instructions what
# to do. However, we have not loaded the package namespace - we access all
# of the functions directly with the msa:: prefix. This method breaks down
# when generic functions are involved. I.e. - we could make it work, but
# the amount of code we need then is unreasonable. The straightforward
# way is to load the package. We can still use the prefix notation for
# its functions, just to emphasize where the function comes from. But since
# the namespace then exists, we ensure that generics are properly dispatched.
library(msa) # load the msa package namespace
msaT <- new("MsaAAMultipleAlignment", # create new MsaAAMultipleAlignment object
unmasked = x, # "unmasked" slot takes an AAStringSet
version = "T-Coffee", # "version" slot takes a string
params = list(), # "params" takes a list(), we leave the
# list empty, but we could add the
# alignment parameters that we used at
# the EBI here.
call = "imported from T-coffee alignment") # also a string
str(msaT)
msaT # Now we have fabricated an msaAAMultipleAlignment object, and we can
# use the msa package functions on it
msaTScores <- msa::msaConservationScore(msaT, substitutionMatrix = BLOSUM62)
# == 4.2 More alignments ===================================================
# Next, we calculate alignments with msa's two other alignment options:
# CLUSTAL Omega
(msaO <- msa::msaClustalOmega( MBP1set, order = "aligned"))
msaOScores <- msa::msaConservationScore(msaO, substitutionMatrix = BLOSUM62)
# CLUSTAL W
(msaW <- msa::msaClustalW( MBP1set, order = "aligned"))
msaWScores <- msa::msaConservationScore(msaW, substitutionMatrix = BLOSUM62)
# == 4.3 Computing comparison metrics ======================================
# Ready to compare.
# ... sum of alignment scores of alignment divided by sum of alignment scores
# of reference alignment (arbitrarily using CLUSTAL W as reference)
sRef <- sum(msaWScores)
sum(msaWScores) / sRef # CLUSTAl W
sum(msaOScores) / sRef # CLUSTAL O
sum(msaTScores) / sRef # T-COFFEE
sum(msaMScores) / sRef # MUSCLE
# ... mean alignment scores (higher is better)
mean(msaWScores) # CLUSTAl W
mean(msaOScores) # CLUSTAL O
mean(msaTScores) # T-COFFEE
mean(msaMScores) # MUSCLE
# total number of gaps (lower is better)
countGaps <- function(ali) {
x <- paste0(as.character(ali), collapse = "")
aa <- nchar(gsub("-", "", x))
return(nchar(x) - aa)
}
countGaps(msaW) # CLUSTAl W
countGaps(msaO) # CLUSTAL O
countGaps(msaT) # T-COFFEE
countGaps(msaM) # MUSCLE
# number of indels in alignment (lower is less fragmented)
countIndels <- function(ali) {
x <- paste0(as.character(ali), collapse = "") # collapse into single string
x <- unlist(strsplit(x, "")) # split into characters
x <- x == "-" # convert into boolean
x <- rle(x) # calculate rle
# every run of TRUE is one indel event
return(sum(x$values))
}
countIndels(msaW) # CLUSTAl W
countIndels(msaO) # CLUSTAL O
countIndels(msaT) # T-COFFEE
countIndels(msaM) # MUSCLE
# Let's look at the distribution of alignment scores:
boxplot(list(CLUSTAL.W = msaWScores,
CLUSTAL.O = msaOScores,
T.COFFEE = msaTScores,
MUSCLE = msaMScores),
cex.axis = 0.8,
col = c("#7D556488", "#74628F88", "#5E78A188", "#3DAEB288"))
# CLUSTAL W and CLUSTAL O don't look all that different. T-Coffee tends to have
# a tighter distribution with less negative scores. Muscle has a slightly higher
# mean and generally higher scores.
# Boxplots are convenient, but don't give us much detail about the shape of the
# distribution. For that, we need histograms, or density plots.
plot(density(msaWScores),
type = "l",
col = "#7D5564",
lwd = 1.5,
ylim = c(0, (max(density(msaWScores)$y) * 1.3)),
main = "Comparing MSA algorithms",
xlab = "Alignment Score",
ylab = "Density")
points(density(msaOScores),
type = "l",
lwd = 1.5,
col = "#74628F")
points(density(msaTScores),
type = "l",
lwd = 1.5,
col = "#5E78A1")
points(density(msaMScores),
type = "l",
lwd = 1.5,
col = "#3DAEB2")
legend("topright",
legend = c("MUSCLE", "T-COFFEE", "CLUSTAL O", "CLUSTAL W"),
col = c("#3DAEB2", "#5E78A1", "#74628F", "#7D5564"),
lwd = 2,
cex = 0.7,
bty = "n")
# The density plots confirm in more detail that CLUSTAL W misses some of the
# higher-scoring possibilities, that wherever CLUSTAL O is bad, it is quite bad,
# that T-COFFEE has fewer poorly scoring columns but misses some of the better
# scoring possibilities, and that MUSCLE appears to do best overall.
# Can we attribute these differences to sections of the alignment in which the
# algorithms did better or worse? Let's plot the scores cumulatively. The
# alignments have different lengths, so we plot the scores on the respective
# fraction of the alignement length.
plot(seq(0, 1, length.out = length(msaWScores)), # x- axis: fraction of length
cumsum(msaWScores),
type = "l",
col = "#7D5564",
lwd = 1.5,
ylim = c(0, max(cumsum(msaMScores))),
main = "Comparing MSA algorithms",
xlab = "Alignment Position",
ylab = "Cumulative Alignment Score")
points(seq(0, 1, length.out = length(msaOScores)), # x- axis: fraction of length
cumsum(msaOScores),
type = "l",
lwd = 1.5,
col = "#74628F")
points(seq(0, 1, length.out = length(msaTScores)), # x- axis: fraction of length
cumsum(msaTScores),
type = "l",
lwd = 1.5,
col = "#5E78A1")
points(seq(0, 1, length.out = length(msaMScores)), # x- axis: fraction of length
cumsum(msaMScores),
type = "l",
lwd = 1.5,
col = "#3DAEB2")
legend("bottomright",
legend = c("MUSCLE", "T-COFFEE", "CLUSTAL O", "CLUSTAL W"),
col = c("#3DAEB2", "#5E78A1", "#74628F", "#7D5564"),
lwd = 2,
cex = 0.7,
bty = "n")
# Your alignment is going to be different from mine, due to the inclusion of
# MYSPE - but what I see is that MUSCLE gives the highest score overall, and
# achieves this with fewer indels than most, and the lowest number of gaps of
# all algorithms.
# To actually compare regions of alignments, we need to align alignments.
# = 5 Profile-Profile alignments ==========================================
# Profile-profile alignments are the most powerful way to pick up distant
# relationships between sequence families. The can be used, for example to build
# a profile from structural superpositions of crystal structures, and then map a
# MSA alignment onto those features. Here we will use profile-profile comparison
# to compare two MSAs with each other, by aligning them. The algorithm is
# provided by the DECIPHER package.
if (! requireNamespace("DECIPHER", quietly=TRUE)) {
BiocManager::install("DECIPHER")
}
# Package information:
# library(help = DECIPHER) # basic information
# browseVignettes("DECIPHER") # available vignettes
# data(package = "DECIPHER") # available datasets
# AlignProfiles() takes two AAStringSets as input. Let's compare the MUSCLE and
# CLUSTAL W alignments: we could do this directly ...
DECIPHER::AlignProfiles(msaW@unmasked, msaM@unmasked)
# But for ease of comparison, we'll reorder the sequences of the CLUSTAL W
# alignment into the same order as the MUSCLE alignment:
m <- as.character(msaM)
w <- as.character(msaW)[names(m)]
(ppa <- DECIPHER::AlignProfiles(AAStringSet(w), AAStringSet(m)))
# Conveniently, AlignProfiles() returns an AAStringSet, so we can use our
# writeALN function to show it. Here is an arbitrary block, from somewhere in
# the middle of the alignment:
writeALN(ppa, range = c(751, 810))
# If you look at this for a while, you can begin to figue out where the
# algorithms made different decisions about where to insert gaps, and how to
# move segments of sequence around. But matters become more clear if we
# post-process this profile-profile alignment. Let's replace all hyphens that
# the pp-alignment has inserted with blanks, and let's add a separator line down
# the middle between the two alignments.
x <- unlist(strsplit(as.character(ppa), "")) # unlist all
dim(x) <- c(width(ppa)[1], length(ppa)) # form into matrix by columns
x <- t(x) # transpose the matrix
(a1 <- 1:(nrow(x)/2)) # rows of alignment 1
(a2 <- ((nrow(x)/2) + 1):nrow(x)) # rows of alignment 2
for (i in 1:ncol(x)) {
if (all(x[a1, i] == "-")) { x[a1, i] <- " " } # blank hyphens that shift
if (all(x[a2, i] == "-")) { x[a2, i] <- " " } # original alignment blocks
}
# collapse the matrix into strings
ppa2 <- character()
for (i in 1:nrow(x)) {
ppa2[i] <- paste0(x[i, ], collapse = "")
names(ppa2)[i] <- names(ppa)[i]
}
# add a separator line
x <- paste0(rep("-", width(ppa)[1]), collapse = "")
ppa2 <- c(ppa2[a1], x, ppa2[a2])
# inspect
writeALN(ppa2, range = c(800, 960))
# Again, go explore, and get a sense of what's going on. You may find that
# CLUSTAL W has a tendency to insert short gaps all over the alignment, whereas
# MUSCLE keeps indels in blocks. CLUSTAL's behaviour is exactly what I would
# expect from an algorithm that builds alignments incrementally from pairwise
# local alignments, without global refinement.
# = 6 Sequence Logos ======================================================
# To visualize the information that we can get about structure and function with
# an MSA, we'll calculate a sequence logo of the Mbp1 recognition helix - the
# part of the structure that inserts into the major groove of the DNA and
# provides sequence specificity to the DNA binding of this transcription factor.
# Helix-B in Mbp1 with four residues upstream and downstream spans the sequence
# 43-ANFAKAKRTRILEKEVLKE-61
# == 6.1 Subsetting an alignment by motif ==================================
# Finding the location of such an substring in an alignment is not entirely
# trivial, because the alignment might have produced indels in that sequence.
# Our strategy can be:
# - fetch the sequence from the alignment
# - remove all hyphens
# - find the range where the target sequence matches
# - count how many characters in all there are in the aligned sequence, up
# to the start and end of the match
# - these numbers define the range of the match in the alignment.
x <- as.character(msaM)["MBP1_SACCE"]
xAA <- gsub("-", "", x)
motif <- "ANFAKAKRTRILEKEVLKE"
(m <- regexpr(motif, xAA)) # matched in position 43, with a length of 19
(motifStart <- as.numeric(m))
(motifEnd <- attr(m, "match.length") + motifStart - 1)
# To count characters, we split the string into single characters ...
x <- unlist(strsplit(x, ""))
# ... convert this into a boolean, which is true if the character is not
# a hyphen ...
x <- x != "-"
# ... cast this into a numeric, which turns TRUE into 1 and FALSE into 0 ...
x <- as.numeric(x)
# ... and sum up the cumulative sum.
x <- cumsum(x)
# Now we can find where the 43'd and 61'st characters are located in the
# alignment string ...
(aliStart <- which(x == motifStart)[1]) # get the first hit if there are more
(aliEnd <- which(x == motifEnd)[1])
# ... and subset the alignment
(motifAli <- subseq(msaM@unmasked, start = aliStart, end = aliEnd))
# Packaging this into a function is convenient to have, therefore I have added
# such a function to the .utilities.R script: fetchMSAmotif(). Try it:
wing <- "HEKVQGGFGKYQGTWV" # the MBP1_SACCE APSES "wing" sequence
writeALN(fetchMSAmotif(msaM, wing))
# == 6.2 Plot a Sequence Logo ==============================================
# The Bioconductor seqLogo:: packager handles only DNA sequences, but there are
# now several good options to plot sequence logos in R, these include dagLogo,
# DiffLogo, Logolas, and motifStack. For our example we will use ggseqlogo
# written by by Omar Waghi, a former UofT BCB student who is now at the EBI.
if (! requireNamespace("ggseqlogo", quietly=TRUE)) {
install.packages(("ggseqlogo"))
}
# Package information:
# library(help=ggseqlogo) # basic information
# browseVignettes("ggseqlogo") # available vignettes
# data(package = "ggseqlogo") # available datasets
ggseqlogo::ggseqlogo(as.character(motifAli))
# [END]
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