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https://github.com/adamtaranto/corset-tools

Companion scripts for annotation of Corset generated transcript clusters.
https://github.com/adamtaranto/corset-tools

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Companion scripts for annotation of Corset generated transcript clusters.

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README 

        
#Corset-tools

Companion scripts for working with transcript clusters generated by Corset.



##Fetch Cluster Sequences

Given a list of clusters of interest, a transcript fasta, and a Corset mapping file,

**fetchClusterSeqs.py** will return a fasta file of transcripts belonging to those 

clusters (with cluster names appended).



This script is intended for use, following differential expression analysis, to

extract and annotate transcript belonging to DE clusters.



###Usage

fetchClusterSeqs.py -i transcripts.fa -t myfavClusters.csv -o transcriptsWithclusterTags.fa -c clusters.txt



###Options

-i, --inFasta         Multi fasta to extract subset from.



-t, --targetClust     Comma delimited file with target cluster names in column one.



-c, --clustMap        Corset transcript-to-cluster mapping file.



-h, --help            Show help message and exit.



-o, --outFasta        Directory for new sequence file to be written to. i.e. output/newfile.fa



-l  --longest         If set only report the longest transcript per cluster



## Cross Cluster Counter

When using Corset to cluster transcriptomes from two species/isolates/cultivars/etc.,

sequence variation is likely to exist between transcriptomes and it is important to check 

that your Corset clustering has not excessively split clusters based on transcriptome set.



The **crossClustCount.py** script will take a corset cluster mapping (transcript --> cluster),

as well as fasta files for those transcriptomes, and calculate the number of members from 

each source transcriptome in each cluster.



Beware of clusters that only contain transcripts from one transcript set when running 

differential expression analyses - these may create a false differential expression signal.



If you have many clusters in which reads from only one transcriptome set are present, you

might consider adjusting your --score-min threshold in Bowtie2 to be more tolerant of 

variation when mapping reads between transcriptomes. 



###Usage

crossClustCount.py -X transcripts_X.fa -Y transcripts_Y.fa -x SetX_Label -y SetY_Label -o outfile.tab -c clusters.txt



###Arguments

-h, --help          Show help message and exit.



-X, --transFastaX   Fasta file for transcriptome X.



-Y, --transFastaY   Fasta file for transcriptome Y.



-x, --transNameX    Unique lable for transcriptome X.



-y, --transNameY    Unique lable for transcriptome Y.



-o, --outFile       Location for summary file to be written to.



-c, --clustMap      Corset transcript-to-cluster mapping file.



## Transcriptome Distance Calculator 

When using Corset to cluster transcriptomes from two different isolates/individuals/cultivars/species

natural sequence variation is likely to occur between input datasets. For equivalent transcripts

to cluster correctly it is essential that reads are able to cross-map to their eqivalent location 

in the non-self transcriptome.



In Bowtie2, the minimum score for a 'valid' read alignment is set via the --score-min option.

The format is [type,intercept,slope], with the default setting -L,-0.6,-0.6.



The equation for a 100 bp read would be -0.6 + (-0.6 * 100) = -60.6



The default Bowtie 2 penalty for a mismatched base is -6. An alignment gap of length 2 receives a 

penalty of -11 by default (-5 for the gap open, -3 for the first extension, -3 for the second extension). 



Therefore a 100bp read alignment is still considered valid with 5 mismatches (-5 * 5 = -30), 

or one gap + one mismatch (-6 + (-5 + -3) = -14).



Failing to set a minimum score threshold that is below (more negative than) the expected distance between

homologous transcripts will likely result in transcript clusters that split by source transcriptome; or if

homologous clusters are predicted, lower mapping efficiency between sets may create a false differential

expression signal in later analyses (genes that are more variable between isolate two isolates may appear 

to be differentially expressed when exposed to the same treatment condition.)



Conversely, setting --score-min to high will likely result in excessive collapse of paralogues.



The **Transcriptome Distance Calculator** compares equivalent transcript pairs from two transcriptomes

and determines the distance (gaps and mismatches) for each pair, and whether mapping would be successful 

for a given read length and --score-min setting.



Transcript pairs may be provided as a tab delimited list; alternatively, the script can perform a reciprocal 

best blast analysis if provided with tab formated blast results for SetA vs SetB, and SetB vs SetA.



*Pending feature*: Advise user of the minimum alignment score that must be tolerated in order to allow

cross-mapping for 95% of the transcript population.



###Usage

Pairs provided:

transDistCalc.py -r 100 -i -0.6 -s -0.6 -n pairnames.txt -a FASTA_A.fa -b FASTA_B.fa -g -5

			 -e -3 -m -6 -o alignmentreports.txt



Get pairs from reciprocal best blast:

transDistCalc.py -r 100 -i -0.6 -s -0.6 -x BLAST_AvsB.tab -y BLAST_BvsA.tab -a FASTA_A.fa -b FASTA_B.fa -g -5

			 -e -3 -m -6 -o alignmentreports.txt -w



###Options

  -h, --help              Show help message and exit.



  -r, --readLength        Length of reads intended for mapping to transcriptomes.

                          (after quality trimming).



  -i, --scoreMinIntercept Intercept value for Bowtie2 --score-min function.



  -s, --scoreMinSlope     Slope value for Bowtie2 --score-min function.



  -n, --pairNames         Two column tab delimited file containing names for

                          sequences from fastaA and fastaB to be aligned.



  -x, --blastAvB          Blast tab result file for fastaA query against fastaB

                          subject.



  -y, --blastBvA          Blast tab result file for fastaB query against fastaA

                          subject.



  -a, --fastaA            Multifasta set A.



  -b, --fastaB            Multifasta set B.



  -g, --gapOpen           Penalty for opening a gap.



  -e, --gapExtend         Penalty for extending a gap.



  -m, --mismatch          Pentaly for a mismatched position.

  

  -o, --outFile           Write output table to file.



  -w, --recipFile         Write reciprocal blast pairs to file. True if flag is

                          set.