wenmm / splice

SPLICE

Analysis pipeline for long-read RNA-seq data using Nanopore technology

Requirement

• python3
• minimap2 (v2.17 or higher)

Input file

FASTQ file

Usage

Step 1: Preparation of the reference transcriptome file

Download the reference transcriptome file from the UCSC genome browser (https://genome.ucsc.edu/)

1. Select Table Browser from the Tools tab.
2. Select "Genes and Gene Predictions" in the group section.
3. Select the desired database in the Track section and download the file.

Numbering the exons of the reference transcriptome.
If you use files from two databases (e.g. GENCODE and RefSeq), sort them by gene name and transcript name, and remove redundant transcripts.

$ cd <path to SPLICE>
$ sh ref_exonnum.sh <path to reference transcriptome> <path to reference genome sequence (FASTA)> <output directory> 

Edit the configure file

• The path to the reference genome file (FASTA) should be specified on the REF_GENOME_FA line
• The path to the output .exonnum file should be specified on REF_TRANSCRIPT line
• The path to the output .exonnum.fa file should be specified on REF_TRANSCRIPT_FA line
• The path to the minimap2 execution file should be specified on MINIMAP2 line

Step 2: Annotation to reference transcriptome

$ sh SPLICE_annot.sh <path to FASTQ> <output directory>

Step 3: Analysis of expression levels (Option for multiple analysis)

Move all .annot and .novel_exon files to single directory

$ sh SPLICE_exp.sh <output directory of Step2 or directory of `.annot` and `.novel_exon` files (multi-sample analysis)> <output directory>

Output

expression.tsv - Output file of transcript expression levels (number of supporting reads)

gene	transcript	known/novel	coding/non-coding	transcript length	novel information	sample1	sample2	sampleN
GAPDH	NM_001256799.2	known	coding	full	-	71	50	31
CSDE1	NM_001242891.1	known	coding	partial	-	346	40	88
SAP18	-	novel_exon_length	coding	-	*,6,8,10,*/*,k,l,k,*/21140681,21147186/*,0,0,0,-35,0,0,0,0,*	8	0	9

Notation of novel transcript

.fusion - Output file of fusion transcript expression levels (number of supporting reads) for each sample

Number of reads	Read frequency(%)	GeneA/B	ChrA/B	BreakpointA/B	Read IDs
150	28.571	UQCRFS1/YWHAE	chr19/chr17	29207585-29207585/1364879-1364879	86ada1...
29	7.143	RPS6KA5/TMSB4X	chr14/chrX	91060446-91060446/12977041-12977041	38a936...
12	5.128	TMED10/VPS4B	chr14/chr18	75132140-75132140/63390367-63390367	1e0cbc...

breakpoint indicates the range after merging the neighboring breakpoints

Example

Preparation of the reference transcriptome file

$ git clone https://github.com/hkiyose/SPLICE
$ cd SPLICE
$ sh ref_exonnum.sh <path to reference transcriptome file> <path to reference genome sequence (FASTA)> ./example/ref

Edit the configure file as described above.

Annotation to reference transcriptome.

$ sh SPLICE_annot.sh ./example/fastq/sample1_test.fastq ./example/annot
$ sh SPLICE_annot.sh ./example/fastq/sample2_test.fastq ./example/annot

Analysis of expression levels

$ sh SPLICE_exp.sh ./example/annot ./example/exp

Installation and usage via Docker

Install Docker in your computer, and build a Docker image with the following commands.

$ git clone https://github.com/hkiyose/SPLICE.git
$ cd <path to SPLICE>
$ docker build -t splice .

The following command mounts the host directory containing the data to the container. Refer to Step 1 of Usage to download the reference data.

$ docker run --rm -it \
  -v <path to directory of reference transcriptome and referece genome sequence (FASTA)>:/ref \
  -v <path to directory of sample data(FASTQ)>:/sample_input \
  -v <path to output directory>:/sample_output \
  splice

Then Run according to Usage.

Parameter settings in configuration file

If you want to use different parameters, please change the configuration file.

BQ_FILT - Read quality cutoff. Minimum average base quality score (15)
MIN_SC_LEN - Minimum length of the softclip region to be remapped (60)
MQ_FILT - Mapping quality cutoff (0)
MIN_FUSION_DIST - Minimum distance of each transcript in the fusion transcript (200000)
MAX_FUSION_BP_MERGE - Maximam distance to merge fusion gene breakpoints (5)
MIN_FUSION_READ - Minimum number of support reads for fusion transcripts (1)
MIN_FUSION_FREQ - Minimum frequency of the fusion transcript in the total amount of the gene (%) (0.1)
MQ_FILT_NOVEL_EXON - Mapping quality cutoff of novel exon (1)
MIN_READ_NUM - Minimum number of support reads (3)
MIN_READ_FREQ - Minimum frequency of the transcript in the total amount of the gene (%) (1)
RANGE_SJ_EVA - Maximum change of novel exon length for evaluate the error rate (20)
ERR_RATE_FILT - Mapping error rate cutoff at splicing junctino sites (%) (20)
MIN_NOVEL_LEN_GAP - Minimum change of novel exon length (5)
MIN_NOVEL_EXON_LEN - Minimum length of novel exon (60)

License

GPLv3

Contact

Hiroki Kiyose - [email protected]