Brendel Group Presentation: 21 Nov 2013

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The final word on the P. dominula differential expression analysis.

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Brendel Group Presentation: 21 Nov 2013

  1. 1. Differential gene expression in Polistes dominula Daniel S. Standage, Brendel Group Meeting, 21 Nov 2013 
  2. 2. Context  Basic differential expression analysis  Isoform-level analysis unreliable  Refocused on locus-level DE analysis
  3. 3. Interval loci (iLoci)  Partition genome into segments that contain  0 protein-coding genes  1 protein-coding gene  2 or more overlapping protein-coding genes  P. dominula genome contains 18,675 iLoci  8,531 with 0 genes  9,197 with 1 gene  947 with 2-5 genes
  4. 4. Out-of-the-box analysis  RSEM: estimate expression levels for each sample independently (uses Bowtie to align reads)  Combine expression data into a single matrix  EBSeq: normalize expression levels and identify differentially expressed genes
  5. 5. Results and observations  295 differentially expressed iLoci  Grouping of samples is troubling  Similar concerns as with previous analysis  Some iLoci with very many reads mapped  Some iLoci with very few reads mapped  Concerns about normalizing over such a large dynamic range
  6. 6. Results and observations  294 differentially expressed iLoci  Grouping of samples is troubling  Similar concerns as with previous analysis  Some iLoci with very many reads mapped  Some iLoci with very few reads mapped  Concerns about normalizing over such a large dynamic range
  7. 7. iLocus filtering  Filtered the iLoci based on  Number of reads mapped  Number of samples with reads mapped  Distribution of mapped reads across samples  10,043 / 18,675 iLoci (54%) passed filtering criteria  Re-ran RSEM/EBSeq procedure from scratch
  8. 8. New results  123 differentially expressed iLoci  1 sample (queen 4) still inconsistently grouped
  9. 9. Analysis sans Q4  Removed the Q4 sample and re-ran EBSeq step  Verified normalization is working as we expected  Found very clean result
  10. 10. Analysis sans Q4  Identified 314 differentially expressed iLoci  219 (70%) over-expressed in workers  95 contain 0 genes  197 contain 1 gene  22 contain 2 or more genes
  11. 11. Biological interpretation  Manual analysis of DE iLoci  xGDBvm  yrGATE  Two protein families occurred very frequently  Cytochrome P450s  NADH dehydrogenases
  12. 12. 5 questions  How many CYP genes are in the wasp genome?  What percentage of these CYP genes are DE?  Do CYPs and NADH dehydrogenases belong to the same pathways?  Can the CYP genes in the genome be categorized?  Can reads discarded during genome assembly provide insight into mitochondrial contamination?
  13. 13. CYPs in Polistes dominula  Identified with a basic BLASTP search  Query: translations of Maker annotations  Database: Hymenopteran CYPs from NCBI  154 iLoci potentially contain CYP genes  Not all matched queries represent CYPs  Stricter criteria required for high-confidence count
  14. 14. Differentially expressed CYP genes  Took intersection of 2 lists  mRNAs from DE iLoci  mRNAs potentially encoding CYPs  Identified 12 putative DE CYPs  11 verified manually  9 / 11 over-expressed in queens
  15. 15. DE NADH dehydrogenase genes  BLASTP search found 38 potential NADHdh genes  12-15 DE NADHdh genes  16 putative DE NADHdh genes  1 thrown out by manual examination  3 borderline  14 / 15 are over-expressed in workers

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