The document summarizes Daniel Standage's analysis of differential gene expression in the paper wasp Polistes dominula. He performed locus-level differential expression analysis after isoform-level analysis proved unreliable. The genome contains 18,675 intervals (iLoci) partitioned by gene content. Initial analysis found 295 differentially expressed iLoci but sample grouping was problematic. After filtering iLoci by read counts and re-running the analysis without a problematic sample, 314 differentially expressed iLoci were identified, mostly over-expressed in workers. Cytochrome P450 and NADH dehydrogenase genes were frequently differentially expressed.

1. Differential gene expression in
Polistes dominula
Daniel S. Standage, Brendel Group Meeting, 21 Nov 2013
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2. Context
 Basic differential expression analysis
 Isoform-level analysis unreliable
 Refocused on locus-level DE analysis

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. 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. 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

7. 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

8. 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

9. New results
 123 differentially expressed iLoci
 1 sample (queen 4) still inconsistently grouped

11. Analysis sans Q4
 Removed the Q4 sample and re-ran EBSeq step
 Verified normalization is working as we expected
 Found very clean result

14. 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

16. Biological interpretation
 Manual analysis of DE iLoci
 xGDBvm
 yrGATE
 Two protein families occurred very frequently
 Cytochrome P450s
 NADH dehydrogenases

17. 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?

18. 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

19. 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

20. 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