Gene Prediction Final Presentation

Transcription

1 Gene Prediction Final Presentation

2 Final Proposed Pipeline Assembled Genome Protein - coding Gene Prediction Ab Initio Prodigal Glimmer GeneMarkS RNA Gene Prediction ncrna Specific trnascanse (trna) RNAmmer (rrna) Infernal Validation BLAST (RefSeq) DEG Merge Validation Final Output

3 Methods for Merging Results Ab initio Tools: GeneMarkS and Prodigal Overlap Criteria: -If there was total overlap, keep the gene -If overlap is not an exact match, then validate via homology 1.Overlap between each pair and between both tools 2.Find overlap between GeneMarkS and Prodigal. Predicted genes with partial or no overlap will then be checked against homology

4 Merging Results from Ab-initio Tools Genes detected by both tools Exact Match Stop Codon Match >60 bp Overlap Genes detected by only one tool Putative Gene List

5 Correcting Previous Results: Reference Genome 2 Tool Exact Match Stop Overlap Extra (FP) Missed (FN) Sensitivity GeneMark- S % 96.7% Prodigal % 98.0% PPV GLIMMER % 92.6% FGenesB % 97.6% Homology % 93.2% Complete Union of Prodigal and GeneMarkS Overlap Union of Prodigal and GeneMarkS % 91.5% % 94.1%

6 Average number of genes predicted by Ab-initio Prodigal tools GMS Prodigal Only* 130 Overlap > 60bp 326 Exact Matches 4257 GMS Only 103

7 Ab initio results

8 Ab initio results

9 Blast Validation Run BLASTx on putative gene list to aid in finding true genes Filtered output Query coverage >95% and Percent identity >95% E-val threshold 1e-10 Putative Genes List: Genes predicted by ab initio programs True positives: Genes verified (by BLASTx) Novel genes: Genes predicted by all ab inito programs but no BLAST hits False positives: Genes predicted by 1 tool but no BLAST hits

10 PREDICTION STATISTICS Sample ID True Positive: Homology G+P NOVEL: Homology G+P False Positive G (or) P OB OB OB OB OB OB OB OB OB OB OB OB Sample ID True Positive: Homology G+P NOVEL: Homology G+P False Positive G (or) P OB OB OB OB OB OB OB OB OB OB OB OB

11 Database for Essential Genes Database contains minimal gene set needed to support cellular life Blast against Database to identify homologs in our predicted genes Salmonella essential genes ~635

12 Sample Predicted Genes Sample Predicted Genes Sample Predicted Genes OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB OB

13 trnascan-se Prediction Results Command: trnascan-se -P../assembly/OB0001.plasmid.scaffolds.fasta awk -f ~/awks/converttrnascantogtf > OB0001.plasmid.scaffolds.gtf Evaluation according to the NCBI Prokaryotic Genome Annotation Pipeline No trnascan-se scores below of 23 assemblies had a trna for each of 21 amino acids plus selenocysteine OB0005 missing Glu OB0006 missing Glu OB0009 missing Glu, His, Trp, and Ile Average number of predicted trnas was 80.6, the minimum was 74, and the maximum was 90

14 RNAmmer Prediction result Command:./rnammer -S bac -multi -gff output.gff < input.fasta -S kingdom bac -multi runs all molecules and both strands in parallel -gff/ -f: Output formats

15 Rfam with Infernal Prediction of all RNAs Command: cmscan --tblout <cmdb> <seqfile> Input: FASTA Output: Infernal specific table format gff Format File {if (substr($0, 0, 1)!= "#") printf("%- 30s infernal%- 20s %- 10s %- 10s %- 8s %- 4s\n", $3, $1, $8, $9, $15, $10); } Command: awk -f File infernal-table-file > output-file.gff Data is merged based on the scoring system of Infernal

16 Infernal Prediction Result

17 Merging rrna, trna, and CDS Predictions CDS Overlap rrna? No >30bp Overlap trna? No Keep CDS Yes Yes Discard CDS Pseudo or atypical trna Not hypothetical CDS? No Hypothetical CDS trna neither pseudo or atypical? No Yes Ye s Keep Both Keep CDS only Keep trna only

18 ncrna CDS Merging Results Rules: If a protein coding gene overlaps with an rrna gene, it is discarded. If a protein coding gene overlaps by 30 bases or more with a trna gene either may be discarded according to the following: Results: If the protein coding gene is homologous to a CDS gene we consider it a confident prediction. If the trna gene is not a pseudogene we consider it a confident prediction. If either prediction is not a confident prediction, it is discarded, unless both aren t, in which case both are kept 2 protein coding genes from OB0006 removed due to trna conflict 1 protein coding gene from OB0013 removed due to trna conflict

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