Genome Assemblies, Analyses, and Comparisons

National Library Of Medicine

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Abstract

Detection and removal of foreign contamination on RNA-Seq samples: Our transcriptome assembly and annotation pipeline include a workflow to detect and remove foreign RNA contamination from the input samples. RNA-Seq contamination has played a large role in misleading multiple research conclusions. It is most troublesome if the target organism does not have a reference genome or annotation in public databases. We have developed GTax, a taxonomic structured database of genomic sequences that can be used with BLAST for taxonomic classification and contamination filtering. This approach efficiently detects and eliminates contaminant reads in RNA-Seq data. GTax genomic sequences were extracted from the NCBI Genome database, using Datasets. The database includes a subset of the latest assemblies of a collection of reference genomes. Sequences were filtered by RefSeq Accession prefixes to reduce the size and possible contaminated sequences. The sequences were organized into 19 mutually exclusive and hierarchical taxonomic groups. For example, taxonomies in the Viridiplantae kingdom are divided into three GTax groups: the Liliopsida group, which includes all monocotyledon sequences; the Eudicotyledons group, which includes all dicotyledon sequences; and the Viridiplantae group, into which all of the other taxa in the Viridiplantae kingdom are placed. The same principle is applied to the Chordata phylum and all taxonomy groups from Neoteleostei to Sarcopterygii. Finally, all remaining Eukaryote taxa are placed in the Eukaryota taxonomy group. This taxonomic structured division of the genomic sequences in GTax keeps phylogenetically closely related species in the same taxonomy group and greatly reduces the size of the searchable BLAST database. The Sauropsida group, which is the biggest group and contains 1,073 sequences and 46,172,754,879 total bases, is only 6.84% of the NT database. Current version of GTax sequences represent 72.18% of the NT database. Our decontamination approach is initiated with a screening of the RNA-Seq reads (using BLAST) against the taxonomy group of the target organism. In these cases, we can screen millions of RNA-Seq reads against less than 6% of the NT database. Then, unidentified reads are screened against the remainder of the GTax taxonomy groups. Reads labeled as correct are those that match the taxonomy group of the target organism. Those that remain unidentified are labeled as such. We use a de novo transcriptome assembly of Solanum lycopersicum (tomato) to demonstrate that removing foreign contamination in sequencing samples reduces the number of assembled chimeric transcripts.