Lorax: A long-read analysis toolbox for cancer and population genomics

In cancer genomics, long-read de novo assembly approaches may not be applicable because of tumor heterogeneity, normal cell contamination and aneuploid chromosomes. Generating sufficiently high coverage for each derivative, potentially sub-clonal, chromosome is not feasible. Lorax is a targeted approach to reveal complex cancer genomic structures such as telomere fusions, templated insertions or chromothripsis rearrangements. Lorax is NOT a long-read SV caller, this functionality is implemented in delly.

Installing lorax

Lorax is available as a statically linked binary, a singularity container (SIF file) or as a docker container. You can also build lorax from source using a recursive clone and make. Lorax depends on HTSlib and Boost.

git clone --recursive https://github.com/tobiasrausch/lorax.git

cd lorax/

make all

Linear reference genomes

Lorax has several subcommands for alignments to linear reference genomes.

Templated insertion threads

Templated insertions threads can be identified using

lorax tithreads -g hg38.fa -o tithreads.bed -m control.bam tumor.bam

The out.bed file specifies nodes (templated insertion source sequences) and edges (templated insertion adjacencies) of a graph that can be plotted using dot.

cut -f 4,9 out.bed | sed -e '1s/^/graph {\n/' | sed -e '$a}' > out.dot

dot -Tpdf out.dot -o out.pdf

The out.reads file lists unique assignments of reads to templated insertion source sequences. To extract the FASTA sequences for all these reads use the lorax extract subcommand (below) with the -a option.

tail -n +2 out.reads | cut -f 1 | sort | uniq > reads.lst

lorax extract -a -g hg38.fa -r reads.lst tumor.bam

Telomere repeats associated with complex rearrangements

Telomere-associated SVs can be identified with lorax using

lorax telomere -g t2t.fa -o outprefix tumor.bam

The output files cluster reads into distinct telomere junctions that can be locally assembled. Since telomeres are repetitive, common mis-mapping artifacts found in a panel of normal samples are provided in the maps subdirectory. It is recommended to use the telomere-to-telomere assembly as the reference genome for lorax telomere.

Read selection for targeted assembly of amplicons

Given a list of amplicon regions and a phased VCF file, lorax can be used to extract amplicon reads for targeted assembly approaches.

lorax amplicon -g hg38.fa -s sample -v phased.bcf -b amplicons.bed tumor.bam

The amplicon subcommand outputs the selected reads (as a hash list out.reads) and a diagnostic table (out.bed) with amplicon regions and their support by split-reads. Ideally, all amplicon regions are connected and belong to one connected component (one cluster of amplicons). This amplicon graph can be plotted using dot.

cut -f 4,11 out.bed | sed -e '1s/^/graph {\n/' | sed -e '$a}' > out.dot

dot -Tpdf out.dot -o out.pdf

To extract the FASTA sequences for all reads use the lorax extract subcommand (below) with the -a option.

Extracting pairwise matches and FASTA sequences of reads

To get FASTA sequences and pairwise read to genome matches for a list of reads (list.reads) use

lorax extract -g hg38.fa -r list.reads tumor.bam

If the read list contains hashes instead of read names as from the lorax amplicon subcommand then please use the -a command-line option.

lorax extract -a -g hg38.fa -r list.reads tumor.bam

Pan-genome graphs

For pan-genome graphs and pan-genome graph alignments, lorax supports the below subcommands, some are work-in-progress.

Connected components of a pan-genome graph

lorax components pangenome.gfa.gz > comp.tsv

Converting a pan-genome (sub-)graph to dot format

lorax gfa2dot -s s103 -r 3 pangenome.gfa.gz > graph.dot

dot -Tpng graph.dot > graph.png

Converting pan-genome graph alignments to BAM

With long reads aligned to a pan-genome graph

minigraph --vc -cx lr pangenome.gfa.gz input.fastq.gz | bgzip > sample.gaf.gz

lorax can be used to convert the graph alignment to BAM

lorax convert -g pangenome.gfa.gz -f input.fastq.gz sample.gaf.gz | samtools sort -o sample.bam -

Node coverage of pan-genome graph alignments

lorax ncov -g pangenome.gfa.gz sample.gaf.gz > ncov.tsv

Citation

Tobias Rausch, Rene Snajder, Adrien Leger, Milena Simovic, Mădălina Giurgiu, Laura Villacorta, Anton G. Henssen, Stefan Fröhling, Oliver Stegle, Ewan Birney, Marc Jan Bonder, Aurelie Ernst, Jan O. Korbel
Long-read sequencing of diagnosis and post-therapy medulloblastoma reveals complex rearrangement patterns and epigenetic signatures
Cell Genomics, 2023, 100281, DOI: 10.1016/j.xgen.2023.100281

License

Lorax is distributed under the BSD 3-Clause license. Consult the accompanying LICENSE file for more details.