![Experimental design
6 queen samples
6 worker samples
Queen x and worker x from same colony
(x ∈ [1 .. 6])
Hypothesis: identify handful of critical
caste-related genes/transcripts](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)
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Brendel Group Presentation: 17 Oct 2013
- 1. Differential expression in the paper wasp Polistes dominula Daniel S. Standage, Brendel Group Meeting, 17 Oct 2013
- 2. Experimental design 6 queen samples 6 worker samples Queen x and worker x from same colony (x ∈ [1 .. 6]) Hypothesis: identify handful of critical caste-related genes/transcripts
- 3. Initial (naïve) analysis with RSEM/EBSeq 209,675 transcripts (assembled by Trinity) RSEM and EBSeq completed without warnings 80-85% reads mapped Many DE transcripts reported 5,769 (FDR=.05) 4,763 (FDR=.01) 3,878 (FDR=.001)
- 4. Permutation testing Randomly shuffle caste labels (queen or worker) Re-run differential expression analysis Repeat test Compare number of transcripts reported as DE for each permutation https://github.com/standage/dept
- 5. Permutation testing Real data 4,763 (FDR=.01) Permutation 1 5,112 Permutation 2 4,174 Permutation 3 4,474 Permutation 4 4,307 Permutation 5 4,718 Permutation 6 4,312 Permutation 7 4,171 Permutation 8 4,714 Permutation 9 3,828 Permutation 10 5,192
- 6. Some observations Some expression levels very low Some transcripts had very few reads mapped Some transcripts had many read mapped Difficulty normalizing over large dynamic range?
- 7. Filter transcripts Reads mapped queen/worker reads mapped > 2,500 overall reads < 1,000,000 Samples 4+ queen/worker samples with > 0 reads mapped Distribution of reads mapped mean(queen/worker reads mapped) * 0.9 > stdev(queen/worker reads mapped)
- 8. DE analysis on filtered transcripts 40,498 transcripts RSEM/EBSeq completed without warnings 20-35% reads mapped Still many DE transcripts reported 1,680 (FDR=.05) 1,328 (FDR=.01) 1,037 (FDR=.001)
- 9. PdomTSAr1.1-034114 (FC=126) Sample Expression Reads mapped Reads (adjusted) Q1 0.00 5232 5669.09 Q2 0.00 10046 5148.89 Q3 51.18 9188 6644.97 Q4 136.68 7920 6901.36 Q5 698.51 27862 6712.76 Q6 0.00 2582 5739.05 W1 0.00 5866 6920.72 W2 0.00 2046 5029.50 W3 0.00 2628 5879.19 W4 0.00 4308 5022.74 W5 0.00 7396 5983.82 W6 0.00 9132 6467.88
- 10. PdomTSAr1.1-007723 (FC=2) Sample Expression Reads mapped Reads (adjusted) Q1 198.82 928 1005.53 Q2 445.48 1864 955.36 Q3 335.03 1330 961.89 Q4 267.42 1048 913.21 Q5 908.57 3988 960.82 Q6 114.54 458 1018.00 W1 125.65 714 842.38 W2 0.00 318 781.71 W3 78.41 426 953.02 W4 116.07 650 757.84 W5 161.56 1028 831.72 W6 147.01 1262 893.83
- 11. RSEM expected count 'expected_count' is the sum of the posterior probability of each read comes from this transcript over all reads. Because 1) each read aligning to this transcript has a probability of being generated from background noise; 2) RSEM may filter some alignable low quality reads, the sum of expected counts for all transcript are generally less than the total number of reads aligned.
- 12. Next (final) steps Look more into “expected count” Additional filtering? Publish!