LongTron: Automated Analysis of Long Read Spliced Alignment Accuracy

MotivationLong read sequencing has increased the accuracy and completeness of assemblies of various organisms genomes in recent months. Similarly, spliced alignments of long read RNA sequencing hold the promise of delivering much longer transcripts of existing and novel isoforms in known genes without the need for error-prone transcript assemblies from short reads. However, low coverage and high-error rates potentially hamper the widespread adoption of long-read spliced alignments in annotation updates and isoform-level expression quantifications. ResultsAddressing these issues, we first develop a simulation of error modes for both Oxford Nanopore and PacBio CCS spliced-alignments. Based on this we train a Random Forest classifier to assign new long-read alignments to one of two error categories, a novel category, or label them as non-error. We use this classifier to label reads from the spliced-alignments of the popular aligner minimap2, run on three long read sequencing datasets, including NA12878 from Oxford Nanopore and PacBio CCS, as well as a PacBio SKBR3 cancer cell line. Finally, we compare the intron chains of the three long read alignments against individual splice sites, short read assemblies, and the output from the FLAIR pipeline on the same samples. Our results demonstrate a substantial lack of precision in determining exact splice sites for long reads during alignment on both platforms while showing some benefit from postprocessing. This work motivates the need for both better aligners and additional post-alignment processing to adjust incorrectly called putative splice-sites and clarify novel transcripts support. Availability and implementationSource code for the random forest implemented in python is available at https://github.com/schatzlab/LongTron under the MIT license. The modified version of GffCompare used to construct Table 3 and related is here: https://github.com/ChristopherWilks/gffcompare/releases/tag/0.11.2LT O_TBL View this table: org.highwire.dtl.DTLVardef@117255borg.highwire.dtl.DTLVardef@95a815org.highwire.dtl.DTLVardef@14e21faorg.highwire.dtl.DTLVardef@98e040org.highwire.dtl.DTLVardef@aab93e_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 3.C_FLOATNO O_TABLECAPTIONIsoform comparison table, using gene models from Gencode V29, plus the isoforms from all the union of annotations; both exact and fuzz comparisons of the set of long-read derived isoforms which 1) match in number of introns or 2) are contained or contain a reference isoform. Percentages use the "Total Intron Chains" as the denominator for the row. C_TABLECAPTION C_TBL Supplementary InformationSupplementary notes and figures are available online.