291 lines
10 KiB
R
291 lines
10 KiB
R
# BIN-ALI-Optimal_sequence_alignment.R
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#
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# Purpose: A Bioinformatics Course:
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# R code accompanying the BIN-ALI-Optimal_sequence_alignment unit.
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#
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# Version: 0.1
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#
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# Date: 2017 08 28
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# Author: Boris Steipe (boris.steipe@utoronto.ca)
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#
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# Versions:
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# 0.1 First code copied from 2016 material.
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#
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# TODO:
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#
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#
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# == DO NOT SIMPLY source() THIS FILE! =======================================
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# If there are portions you don't understand, use R's help system, Google for an
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# answer, or ask your instructor. Don't continue if you don't understand what's
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# going on. That's not how it works ...
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# ==============================================================================
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# = 1 Biostrings Pairwise Alignment
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# Biostrings is one of the basic packages that the Bioconductor software
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# landscape builds on. It stores sequences in "AAstring" objects and these are
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# complex software structures that are designed to be able to handle
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# genome-scale sequences. Biostrings functions - such as the alignment functions
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# - expect their input to be Biostrings objects.
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?AAString
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AAString("ACDE")
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s <- AAString("ACDE")
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str(s)
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# See: it's complicated. This is an "S4" object. Bioconductor uses these objects
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# almost exclusively, but we will not be poking around in their internals. Just
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# this: how do we get the sequence back out of an AAString object? The help page
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# for XString - the parent "class" of AAStrings - mentions the alternatives:
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as.character(s) # the base R version
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toString(s) # using the Biostrings function toString()
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# While we need to remember to convert our sequences from the character vectors
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# that we store in our database, to AAStrings that we can align, the alignment
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# itself is really straightforward. The pairwiseAlignment() function was written
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# to behave exactly like the functions you encountered on the EMBOSS server.
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# First: make AAString objects ...
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sel <- myDB$protein$name == "MBP1_SACCE"
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aaMBP1_SACCE <- AAString(myDB$protein$sequence[sel])
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sel <- myDB$protein$name == paste("MBP1_", biCode(MYSPE), sep = "")
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aaMBP1_MYSPE <- AAString(myDB$protein$sequence[sel])
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?pairwiseAlignment
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# ... and align.
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# Global optimal alignment with end-gap penalties is default. (like EMBOSS needle)
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ali1 <- pairwiseAlignment(
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aaMBP1_SACCE,
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aaMBP1_MYSPE,
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substitutionMatrix = "BLOSUM62",
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gapOpening = 10,
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gapExtension = 0.5)
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str(ali1) # Did you think the AAString object was complicated ?
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# This is a Biostrings alignment object. But we can use Biostrings functions to
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# tame it:
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ali1
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writePairwiseAlignments(ali1) # That should look familiar
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# And we can make the internal structure work for us (@ is for classes as
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# $ is for lists ...)
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str(ali1@pattern)
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ali1@pattern
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ali1@pattern@range
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ali1@pattern@indel
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ali1@pattern@mismatch
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# or work with "normal" R functions
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# the alignment length
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nchar(ali1@pattern)
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# the number of identities
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sum(s2c(as.character(ali1@pattern)) ==
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s2c(as.character(ali1@subject)))
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# ... e.g. to calculate the percentage of identities
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100 *
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sum(s2c(as.character(ali1@pattern)) ==
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s2c(as.character(ali1@subject))) /
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nchar(ali1@pattern)
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# ... which should be the same as reported in the writePairwiseAlignments()
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# output. Awkward to type? Then it calls for a function:
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#
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percentID <- function(al) {
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# returns the percent-identity of a Biostrings alignment object
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return(100 *
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sum(s2c(as.character(al@pattern)) ==
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s2c(as.character(al@subject))) /
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nchar(al@pattern))
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}
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percentID(ali1)
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# Compare with local optimal alignment (like EMBOSS Water)
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ali2 <- pairwiseAlignment(
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aaMBP1_SACCE,
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aaMBP1_MYSPE,
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type = "local",
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substitutionMatrix = "BLOSUM62",
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gapOpening = 50,
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gapExtension = 10)
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writePairwiseAlignments(ali2) # This has probably only aligned the N-terminal
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# DNA binding domain - but that one has quite
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# high sequence identity:
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percentID(ali2)
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# == TASK: ==
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# Compare the two alignments. I have weighted the local alignment heavily
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# towards an ungapped alignment by setting very high gap penalties. Try changing
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# the gap penalties and see what happens: how does the number of indels change,
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# how does the length of indels change...
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# Fine. Please return to the Wiki to study BLAST alignment...
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# ==============================================================================
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# PART FOUR: APSES Domain annotation by alignment
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# ==============================================================================
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# In this section we define the MYSPE APSES sequence by performing a global,
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# optimal sequence alignment of the yeast domain with the full length protein
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# sequence of the protein that was the most similar to the yeast APSES domain.
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#
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# I have annotated the yeast APSES domain as a proteinAnnotation in the
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# database. To view the annotation, we can retrieve it via the proteinID and
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# featureID. Here is the yeast protein ID:
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myDB$protein$ID[myDB$protein$name == "MBP1_SACCE"]
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# ... assign it for convenience:
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proID <- myDB$protein$ID[myDB$protein$name == "MBP1_SACCE"]
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# ... and if you look at the feature table, you can identify the feature ID
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myDB$feature[ , c("ID", "name", "description")]
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myDB$feature$ID[myDB$feature$name == "APSES fold"]
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# ... assign it for convenience:
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ftrID <- myDB$feature$ID[myDB$feature$name == "APSES fold"]
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# ... and with the two annotations we can pull the entry from the protein
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# annotation table
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myDB$proteinAnnotation[myDB$proteinAnnotation$protein.ID == proID &
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myDB$proteinAnnotation$feature.ID == ftrID, ]
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myDB$proteinAnnotation$ID[myDB$proteinAnnotation$protein.ID == proID &
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myDB$proteinAnnotation$feature.ID == ftrID]
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# ... assign it for convenience:
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fanID <- myDB$proteinAnnotation$ID[myDB$proteinAnnotation$protein.ID == proID &
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myDB$proteinAnnotation$feature.ID == ftrID]
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# The annotation record contains the start and end coordinates which we can use
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# to define the APSES domain sequence with a substr() expression.
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substr(myDB$protein$sequence[myDB$protein$ID == proID],
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myDB$proteinAnnotation$start[myDB$proteinAnnotation$ID == fanID],
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myDB$proteinAnnotation$end[myDB$proteinAnnotation$ID == fanID])
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# Lots of code. But don't get lost. Let's recapitulate what we have done: we
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# have selected from the sequence column of the protein table the sequence whose
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# name is "MBP1_SACCE", and selected from the proteinAnnotation table the start
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# and end coordinates of the annotation that joins an "APSES fold" feature with
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# the sequence, and used the start and end coordinates to extract a substring.
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# The expressions get lengthy, but it's not hard to wrap all of this into a
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# function so that we only need to define name and feature.
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dbGetFeatureSequence
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dbGetFeatureSequence(myDB, "MBP1_SACCE", "APSES fold")
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# Let's convert this to an AAstring and assign it:
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aaMB1_SACCE_APSES <- AAString(dbGetFeatureSequence(myDB,
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"MBP1_SACCE",
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"APSES fold"))
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# To align, we need the MYSPE sequence. Here is it's definition again, just
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# in case ...
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sel <- myDB$protein$name == paste("MBP1_", biCode(MYSPE), sep = "")
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aaMBP1_MYSPE <- AAString(myDB$protein$sequence[sel])
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# Now let's align these two sequences of very different length without end-gap
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# penalties using the "overlap" type. "overlap" turns the
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# end-gap penalties off and that is crucially important since
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# the sequences have very different length.
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aliApses <- pairwiseAlignment(
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aaMB1_SACCE_APSES,
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aaMBP1_MYSPE,
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type = "overlap",
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substitutionMatrix = "BLOSUM62",
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gapOpening = 10,
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gapExtension = 0.5)
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# Inspect the result. The aligned sequences should be clearly
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# homologous, and have (almost) no indels. The entire "pattern"
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# sequence from QIYSAR ... to ... KPLFDF should be matched
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# with the "query". Is this correct?
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writePairwiseAlignments(aliApses)
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# If this is correct, you can extract the matched sequence from
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# the alignment object. The syntax is a bit different from what
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# you have seen before: this is an "S4 object", not a list. No
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# worries: as.character() returns a normal string.
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as.character(aliApses@subject)
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# Now, what are the aligned start and end coordinates? You can read them from
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# the output of writePairwiseAlignments(), or you can get them from the range of
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# the match.
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str(aliApses@subject@range)
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# start is:
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aliApses@subject@range@start
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# ... and end is:
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aliApses@subject@range@start + aliApses@subject@range@width - 1
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# Since we have this section defined now, we can create a feature annotation
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# right away and store it in myDB. Copy the code-template below to your
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# myCode.R file, edit it to replace the placeholder items with your data:
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#
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# - The <PROTEIN ID> is to be replaced with the ID of MBP1_MYSPE
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# - The <FEATURE ID> is to be replaced with the ID of "APSES fold"
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# - <START> and <END> are to be replaced with the coordinates you got above
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#
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# Then execute the code and continue below the code template. If you make an
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# error, there are instructions on how to recover, below.
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#
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# ===== begin code template: add a proteinAnnotation to the database =====
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# == edit all placeholder items!
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myProteinID <- "<PROTEIN ID>"
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myFeatureID <- "<FEATURE ID>"
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myStart <- <START>
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myEnd <- <END>
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# == create the proteinAnnotation entry
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panRow <- data.frame(ID = dbAutoincrement(myDB$proteinAnnotation$ID),
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protein.ID = myProteinID,
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feature.ID = myFeatureID,
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start = myStart,
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end = myEnd,
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stringsAsFactors = FALSE)
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myDB$proteinAnnotation <- rbind(myDB$proteinAnnotation, panRow)
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# == check that this was successful and has the right data
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myDB$proteinAnnotation[nrow(myDB$proteinAnnotation), ]
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# ===== end code template ===========================================
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# ... continue here.
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# I expect that a correct result would look something like
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# ID protein.ID feature.ID start end
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# 63 my_fan_1 my_pro_1 ref_ftr_1 6 104
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# If you made a mistake, simply overwrite the current version of myDB by loading
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# your saved, good version: load("myDB.01.RData") and correct your mistake
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# If this is correct, save it
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save(myDB, file = "myDB.02.RData") # Note that it gets a new version number!
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# Done with this part. Copy the sequence of the APSES domain of MBP1_MYSPE - you
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# need it for the reverse BLAST search, and return to the course Wiki.
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# = 1 Tasks
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# [END]
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