FLORA: Fast Long-noncoding RNA Assembly Workflow

FLORA provides easy-to-use command line tools for fast lncRNA transcriptome assembly from RNA-seq BAM files.

Prerequisites

Install bedtools and CPAT. Please make sure bedtools and cpat.py are on the system path.

Installing

Download/Clone the repository and change the working directory to the FLORA directory

python setup.py install

Run

generateFilteredBams.py

Generate BAM files with certain regions removed.

usage: generateFilteredBams [-h] -g INPUTGTF [-t TYPES [TYPES ...]]
                            [-o OUTPUTDIR] [-n NTHREAD]
                            inputBams

positional arguments:
  inputBams             The path to a txt file that contains paths to input
                        bam files separated by the newline character.

optional arguments:
  -h, --help            show this help message and exit
  -g INPUTGTF           The path to reference gene annotation file in GTF
                        format. It can be downloaded from gencode website.
  -t TYPES [TYPES ...]  The gene types to be removed from BAM files.
  -o OUTPUTDIR          Output directory for the output files
  -n NTHREAD            Number of threads to be used for bedtools intersect.

filterTranscripts.py

Identify lncRNA transcripts from transcriptome assembled by Stringtie or Cufflinks.

usage: filterTranscript [-h] -r REFERENCE [-e EXON] [-l LENGTH] [-c CPAT] -x
                        HEXAMER -m LOGIT [-o OUTPUTGTF]
                        inputGTF

positional arguments:
  inputGTF      Input transcriptome file in GTF format. Can be output from
                stringtie or cufflinks

optional arguments:
  -h, --help    show this help message and exit
  -r REFERENCE  The path to reference genome sequences in FASTA format. It
                will be indexed automatically by CPAT if .fai file is not
                present.
  -e EXON       The least number of exons a transcipt should have in order to
                be kept in the final transcriptome
  -l LENGTH     The shortest transcript to be kept in the final transcriptome
  -c CPAT       CPAT cutoff used to filter transcripts
  -x HEXAMER    The path to hexamer table required by CPAT. Can be downloaded
                from CPAT website.
  -m LOGIT      The path to logit model required by CPAT. Can be downloaded
                CPAT website.
  -o OUTPUTGTF  Output prefix for the final transcriptome GTF file

annotateTranscripts.py

Find overlapped and nearby genes of lncRNAs in reference annotation (RefSeq or GENCODE annotation in GFF format)

usage: annotateTranscripts [-h] -r REFERENCE -f PATH [-i IDENTIFIER]
                           [-d DISTANCE] [-n NUMBER] [-o OUTPUT]
                           inputGTF

positional arguments:
  inputGTF       Input transcriptome file in GTF format. The file could be
                 output from Cufflinks or StringTie

optional arguments:
  -h, --help     show this help message and exit
  -r REFERENCE   Identify the source of reference annotation. Currently, it
                 should be either refseq or gencode
  -f PATH        The path to the reference annotation file.
  -i IDENTIFIER  The path to the RefSeq assembly report. The file is used to
                 replace RefSeq sequence identifiers with USCS identifiers.
                 Required if you use RefSeq annotation.
  -d DISTANCE    Specify the distance within which the nearby genes should
                 locate. Default: 10,000 bp. Unit: bp.
  -n NUMBER      Specify the number of nearby genes obtained. Default: 1.
  -o OUTPUT      Output file name for the final annotation. Default:
                 FLORA_annotation.txt