uLTRA implements an alignment method for long RNA sequencing reads based on a novel two-pass collinear chaining algorithm. uLTRA is guided by a database of exon annotations, but it can also be used as a wrapper around minimap2 to align reads outside annotated regions.

References:

• Kristoffer Sahlin and Veli Makinen
  Accurate spliced alignment of long RNA sequencing reads
  Bioinformatics 37(24), Pages 4643–4651 (2021)

• uLTRA GitHub page

• Module Name: ultra (see the modules page for more information)
• Unusual environment variables set
  • ULTRA_HOME  installation directory
  • ULTRA_BIN       executable directory
  • ULTRA_SRC       source code directory
  • ULTRA_DATA  sample data directory

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive --mem=4g
[user@cn0911 ~]$module load ultra   
[+] Loading singularity  3.10.5  on cn0911
[+] Loading Loading ultra  0.1
[user@cn0911 ~]$cp $ULTRA_DATA/* . 
[user@cn0911 ~]$uLTRA pipeline SIRV_genes.fasta SIRV_genes_C_170612a.gtf reads.fa outfolder
creating outfolder
total_flanks2: 90
total_flank_size 72816
total_unique_segment_counter 24396
total_segments_bad 25056
bad 460
total parts size: 24856
total exons size: 74187
min_intron: 20
Number of ref seqs in gff: 71
Number of ref seqs in fasta: 7
Filtering reads aligned to unindexed regions with minimap2
Running minimap2...
minimap2 done.
Done filtering. Reads filtered:0
Time for minimap2:0.10440826416015625 seconds.
Processing reads for NAM finding
Completed processing poly-A tails
Time for processing reads:0.006848812103271484 seconds.
RUNNING NAMFINDER USING COMMAND:
namfinder -k 10 -s 10 -l 10 -u 11 -C 500 -L 1000 -t 3 -S outfolder/refs_sequences.fa outfolder/reads_tmp.fa.gz 2> outfolder/namfinder_stderr.1 | gzip -1 --stdout > outfolder/seeds.txt.gz
Time for namfinder to find seeds:0.48410677909851074 seconds.
Starting aligning reads.
Wrote 0 batches of reads.
file_IO: Reading records done. Tot read: 4
file_IO: Written records in producer process: 0
Process: [0, 0, 0, 0, 0, 0, 0, 0]
Number of instances solved with quadratic collinear chainer solution: 0
Number of instances solved with n*log n collinear chainer solution: 0
Process: [0, 0, 0, 0, 0, 0, 0, 0]
Number of instances solved with quadratic collinear chainer solution: 0
Number of instances solved with n*log n collinear chainer solution: 0
Process: [3.8929521968943623, 4, 0, 0, 0, 0, 0, 0]
Number of instances solved with quadratic collinear chainer solution: 4
Number of instances solved with n*log n collinear chainer solution: 0
Wrote 1 batches of reads.
file_IO: Remainig written records after consumer join: 4
Done joining processes.
Time to align reads:0.07692599296569824 seconds.
Total mm2's primary alignments replaced with uLTRA: 2
Total mm2's alignments unmapped but mapped with uLTRA: 0
2 primary alignments had better score with uLTRA.
0 primary alignments had equal score with alternative aligner.
2 primary alignments had slightly better score with alternative aligner (typically ends bonus giving better scoring in ends, which needs to be implemented in uLTRA).
0 primary alignments had significantly better score with alternative aligner.
0 reads were unmapped with ultra but not by alternative aligner.
0 reads were not attempted to be aligned with ultra (unindexed regions), instead alternative aligner was used.
Time for selecting final best alignments (selecting the best of mm2's vs uLTRA's alignments):0.009923696517944336 seconds.
FSM : 4, NO_SPLICE : 0, Insufficient_junction_coverage_unclassified : 0, ISM/NIC_known : 0, NIC_novel : 0, NNC : 0
total alignment coverage: 3.8929521968943623
Deleting temporary files...
removed: outfolder/minimap2.sam
removed: outfolder/minimap2_errors.1
removed: outfolder/uLTRA_batch_0.stderr
removed: outfolder/uLTRA_batch_1.stderr
removed: outfolder/uLTRA_batch_2.stderr
removed: outfolder/reads_after_genomic_filtering.fasta
removed: outfolder/indexed.sam
removed: outfolder/unindexed.sam
removed: outfolder/refs_sequences.fa
Done.
[user@cn0911 ~]$exit

[user@cn0911 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$