/NCLtk

a toolkit to diagnose ambiguous alignment in circRNA events

Primary LanguageShell

NCLtk

a toolkit to examine ambiguous alignment in non-co-linear trascripts (circular RNAs and trans-splicing)

two modules

1. checking ambiguous alignment in the genomic positions of circRNAs (checkAA_circRNAs.sh):
to concatenate the flanking the circRNAs (within -100 nucleotides to +100 nucleotides for each circRNA's junction) against the reference genome and NCBI Refseq-/GENCODE-/Ensembl- identified mRNAs and ncRNAs using BLAT.
its criteria of ambiguous alignment:
  • alternative co-linear explanation
    to detect alternative co-linear explanation ovelapped more than 90% of the concatenated seq
  • multiple hits
    to mapped to multiple positions with similiar BLAT mapping scores (difference of mapping scores < 5)
2. checking ambiguous alignemnt in the supporting backedspliced junctions' RNA-seqs (checkAA_read.sh):
to concatenate the supporting backspliced junctions' paired-end reads (the concatenated read may be shorter than length of the paired-end reads if the paired-end reads have the overlapped part) against the reference genome and NCBI Refseq-/GENCODE-/Ensembl- identified mRNAs and ncRNAs using BLAT.
its criteria of ambiguous alignment:
  • alternative co-linear explanation
    to detect alternative co-linear explanation ovelapped more than 80% of the concatenated seq
  • multiple hits
    to mapped to multiple positions with similiar BLAT mapping scores (difference of mapping scores < 5)

Citation

Chen C-Y, Chuang T-J (2019) Comment on "A comprehensive overview and evaluation of circular RNA detection tools". PLoS Comput Biol 15(5): e1006158. https://doi.org/10.1371/journal.pcbi.1006158

Chen C-Y, Chuang T-J (2019) NCLcomparator: systematically post-screening non-co-linear transcripts (circular, trans-spliced, or fusion RNAs) identified from various detectors. BMC Bioinformatics 20, 3. https://doi.org/10.1186/s12859-018-2589-0

1. System requirements

The two shell scripts (check_circRNAs.sh and check_reads.sh) runs under Linux-like environment (i.e. Bio-Linux, also see http://environmentalomics.org/bio-linux/) with at least 30 GB RAM.

2. Installation

$ tar zxvf NCLtk.tar.gz
$ cd NCLtk
$ chmod +x *.sh
$ cd bin 
$ chmod +x *

3. Installation external tools

(1) BLAT (http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/blat/)

(2) seqtk (https://github.com/lh3/seqtk)

4. Preparation

(1) Chromosome fasta files

$ mkdir hg19
$ cd hg19
$ wget --timestamping 'ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/chromosomes/*'
$ gunzip *.gz

(2) Genome fasta file

$ wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/GRCh37.p13.genome.fa.gz
$ gunzip GRCh37.p13.genome.fa.gz

(3) Transcritome files

$ mkdir others
$ cd others
$ wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/gencode.v19.pc_transcripts.fa.gz
$ wget ftp://ftp.sanger.ac.uk/pub/gencode/Gencode_human/release_19/gencode.v19.lncRNA_transcripts.fa.gz
$ wget ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/mrna.fa.gz
$ wget ftp://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/refMrna.fa.gz
$ wget ftp://ftp.ensembl.org/pub/grch37/release-91/fasta/homo_sapiens/cdna/Homo_sapiens.GRCh37.cdna.all.fa.gz
$ wget ftp://ftp.ensembl.org/pub/grch37/release-91/fasta/homo_sapiens/ncrna/Homo_sapiens.GRCh37.ncrna.fa.gz
$ gunzip *.gz

5. Execution of checkAA_circRNAs

Usage :

$ ./checkAA_circRNAs.sh -circRNAs [circRNAs.txt] -thread [number of threads] -genome [genome] -genome_chr [chromosome folder] -others [transcriptome folder] -blat [blat link] -tools [bib path]

An example:

$ ./checkAA_circRNAs.sh -circRNAs examples/circ1046.txt -thread 6 -genome /path/to/GRCh37.p13.genome.fa -genome_chr /path/to/hg19/ -others /path/to/others/ -blat /path/to/blat -tools /path/to/NCLtk/bin/

Input format (e.g. circ1046.txt)

circRNA_ID strand
chr1:93297917|93307481 +
chr1:52954581|53019130 -

Output file: ambiguous_align_circ1046.txt

6. Execution of checkAA_reads

Usage :

$ ./checkAA_reads.sh -circReads [circReads.txt] -genome [genome.fa] -others [transcriptome folder] -read1 [read1.fastq or fastq.gz] -read2 [read2.fastq or fastq.gz] -blat [blat link] -seqtk [seqtk link ] -tools [bin path]

An example:

$ ./checkAA_reads.sh -circReads examples/CIRI_positive.txt -genome /path/to/GRCh37.p13.genome.fa -others /path/to/others/ -read1 /path/to/pos_1.fastq.gz -read2 /path/to/pos_2.fastq.gz -blat /path/to/blat -seqtk /path/to/seqtk -tools /path/to/NCLtk/bin/

Input format (e.g. CIRI_positive.txt)

circRNA_ID strand gene_id #junction_reads junction_reads_ID
chr1:8928047|8932045 - ENSG00000074800.9, 3 simulate:21841,simulate:21844,simulate:21846,

Output format (e.g. ambiguous_align_CIRI_positive.txt)

circRNA_ID strand gene_id #junction_reads junction_reads_ID #ambiguous_reads ambiguous_reads_ID
chr1:865535|866469 + ENSG00000187634.6, 6 simulate:69646,simulate:69647,simulate:69648,simulate:69650,simulate:69653,simulate:69657, 0 na
chr1:871152|874509 + ENSG00000187634.6, 5 simulate:69678,simulate:69679,simulate:69682,simulate:69683,simulate:69686, 1 simulate:69679,