RNAxSV : RNAseq isoforms and pathways bounded by evolutionarily conserved SVs

Contributors

1. Evan Biederstedt (Leader, Liaison and Writer)
2. Yutong Qiu (Sysadmin and code developer)
3. Chia Sin Liew (Writer and analyst)
4. Chase Mateusiak (Sysadmin and code developer)
5. Rupesh Kesharwani (Sysadmin and analyst)
6. Bida Gu (Sysadmin and code developer)

Introduction

Gene fusions or transcript fusions are combinations of two genes or transcripts that are created by chromosomal rearrangement and a variety of genomic structural variations (SV), including translocations, inversions, deletions, and tandem duplications (DUP). These chromosomal signatures are distinctive to various cancer types and precision oncology such as in BCR-ABL1. These hybrid genes and/or transcripts can be pedicted using a variety of computational tools and fusion detection techniques. However, when we wish to compare annotation over multiple samples, the lack of comparison tools makes it more difficult.

Goals

The tool aims to compare mulitple annotation (gff and seq) of multiple samples. It provided information about chromosomal rearrnagements (fusions) of genes between the two or more annotations.

Description

The isoseq3 generated HQ (Full-length high quality i.e. accuracy of 99%) transcripts of HG002 (NA27730, NA24385 and NA26105) were mapped to GRCh38 using three different long read alignment tools (uLTRA, deSALT and Minimap2). Next, we performed cDNA_cupcake workflow to collapse the redundant isoforms from bam, followed by filtering the low counts isoforms by 10 and filter away 5' degraded isoforms that might not be biologically significant. And at last, generated corrected gff and fasta as final output which futher utilized as input for the our tool.

Commands:

uLTRA mapping Then the data was processed with uLTRA [ PMID: 34302453 ] (v0.0.4.1; commands: uLTRA align --prefix prefix --isoseq --t 4 --index index_dir/ GRCh38.v33p13.primary_assembly.fa HG002.polished.hq.fastq.gz results_dir/).

deSALT mapping Then the data was processed with deSALT [ PMID: 31842925 ] (v1.5.5; ; commands: deSALT aln -T -o HG002.sam -t 4 -x ccs HG002.polished.hq.fastq.gz).

Minimap2 mapping Then the data was processed with Minimap2 [ PMID: 29750242 ] (v2.24-r1122; commands: minimap2 -t 8 -ax splice:hq -uf --secondary=no -C5 -O6,24 -B4 GRCh38.v33p13.primary_assembly.fa HG002.polished.hq.fastq.gz)

Flowchart

To extract sets of unique isoforms

To annotate the unique isoforms

Quick start

Computational Resources / Operation

References

1. https://agat.readthedocs.io/en/latest/tools/agat_sp_compare_two_annotations.html
2. https://ccb.jhu.edu/software/stringtie/gffcompare.shtml