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LEARNING MATERIALS
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Rationale and Background:

CLASS2: A fast and accurate alternative-splicing aware transcript assembler for RNA-seq reads aligned to a genome.

Song L, Sabunciyan S, Florea L. 2016. Nucleic Acids Res. 44(10):e98.  [ Medline ]

CLASS2 is a fast and accurate program for transcript assembly of RNA-seq reads aligned to a reference genome. It uses the splice graph model to represent a gene and its splice variants, and a dynamic programming optimization algorithm to score and select a subset of transcripts most likely present in the sample. CLASS2 aims to capture as much alternative splicing variation as possible with very high accuracy.

 

Pre-Requisites:

  1. A CyVerse account. (Register for a CyVerse account here - user.cyverse.org)
  2. Inputs
     a. Mandatory
         Input File: one SAM or BAM file
     b. Optional
         -e evidence: the path to the evidence, or annotation, file (GTF format); user can select from the list or use own annotations
         -l label: add a prefix and a "_" to the gene and transcript ids in the output GTF file (default: not used)
         -F isoform_fraction: do not report transcripts whose abundance level is lower than f*|most expressed transcript| in a gene
         --clean: whether to remove the temporary files in -wd (default: yes)
  3. Optional Output File Name
        -o filename: prefix for the output file name

Test/Sample Data

The test data for CLASS2 can be found here: /iplant/home/shared/iplantcollaborative/example_data/class2

Test Run

  1. Open CLASS2 in DE
  2. Choose the input BAM file /iplant/home/shared/iplantcollaborative/example_data/class2/sample.bam
  3. Optionally select the GTF annotation file /iplant/home/shared/iplantcollaborative/example_data/class2/sample.annot.gtf
  4. Optionally set other parameters, such as Isoform fraction (-F) , Prefix for transcript names (-l) Remove depth and splice files (–clean)  
  5. Optionally set the prefix for the output file name
  6. Launch the analysis

Test Results: 

  1. sample.gtf:   Output file
  2. If  selecting 'No' for the '--clean' option, then a folder named "class_tmp" containing alignment, splice junction and read coverage data will be generated. 

The following file illustrates the CLASS2 output for the test data, using the provided annotation:

sample.gtf



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