Samples for RNAseq

One of the problems with past RNAseq studies of imprinting (such as the two Gregg (2010) science papers, see Proudhon (2011), and DeVeal (2012)) was an inadequate sample size and incorrect experimental set-up. This led to a large number of false-positives. To avoid these mistakes I am going to use 40 samples, 10 from each genotype (5 from each sex). Here is the set up:

P. campbelli                         5 female, 5 male
P. campbelli X P. sungorus 5 female, 5 male
P. sungorus X P. campbelli 5 female, 5 male
P. sungorus                          5 female, 5 male

I will be able to sequence 20 samples per lane, meaning that I can't split it perfectly equally between the lanes. In order to avoid batch effects (Auer, 2010), I will use the following set up:

Lane 1:

P. campbelli                          3 female, 2 male
P. campbelli X P. sungorus  3 female, 2 male
P. sungorus X P. campbelli  3 female, 2 male
P. sungorus                           3 female, 2 male



Lane 2:

P. campbelli                          2 female, 3 male
P. campbelli X P. sungorus  2 female, 3 male
P. sungorus X P. campbelli  2 female, 3 male
P. sungorus                           2 female, 3 male



Finally, each of the 5 female and 5 male placentas will be brother-sister pairs, from 5 different litters (this is the ideal, I'll have to do some more sex-typing to see if I can actually make it work - stay tuned. I will definitely assure that the 5 females are from 5 different litters (and the same for the males) but whether all those male-female pairs are brother/sister may be tricky). I am currently debating whether I will have the 3 females in lane 1 sister to the 3 males in lane 2, or whether there should be a mix. I need to go over the Auer (2010) paper again to see if I can glean any more insight from it.

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As for the exact brother-sister pairs:

P. campbelli:
1. BB15.3M    BB15.4F
2. BB77.2M    BB77.1F
3. BB86.2M    BB86.1F
4. no male yet  BB87.1F   ---> male BB87.6M
5. no male yet  BB90.1F   ---> male 77.3M

Average placenta weight of these individuals is: 0.11783g
Overall average of P. campbelli  is:                     0.11122g
quite close-this should be fine.

I need to sex-type more offspring from families BB86 and BB90. There are two more unknowns from 86 and one from 90. Hopefully they will be males, otherwise I'll have to use a brother of 15.1M, 77.2M or 86.2M which would work, but is not ideal.

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P. campbelli X P. sungorus: 
1. BS70.3M     BS70.4F
2. BS72.2M     BS72.1F
3. BS73.3M     BS73.1F
4. BS71.3M     BS70.2F
5. BS11.1M     BS73.2F

You'll notice that for pairs 4 and 5, the individuals are not siblings (what is worse - the females are sibs of pairs 1 and 3) There is nothing else I can do here. The only option would be to set up more crosses. I'll consider this, but I need to do this sequencing sooner rather than later and I don't think that this little bit of my perfectionism is worth the wait. I'll talk to J~ and see what he says.

Average placenta weight of these samples: 0.12916g
Overall average for these hybrids:               0.10948g
Not as close as before, but not awful.
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P. sungorus X P. campbelli:
1. SB20.2M    SB20.6F
2. SB22.4M    SB22.5F
3. SB25.5M    SB25.3F
4. SB29.4M    SB29.1F
5. SB20.8M    SB24.2F

Pair 5 are not sibs, but unless I set up more crosses this is the best I can do.
Average of these samples:             0.47890g
Overall average for these hybrids: 0.39388g
Pretty good.

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P. sungorus:         
1. SS82.1M     SS82.2F
2. SS87.1M     SS87.3F  
3. SS88.1M     SS88.3F
4. SS89.2M     SS89.1F
5. SS91.1M     no female yet ----> female SS91.4F
6. no male yet  SS93.2F        ----> don't use these

I will sex-type the other individuals in SS91 and SS93. If I can find a female from 91 or a male from 93 I will be set. Otherwise, I will use the ones shown above.

Average of these samples:    0.14113
Overall average of SS:         0.14757
Great.

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Now off to sex-type, updates soon...
Here's the results:
lane:                  gender
1 Ladder        
2 BB87.5          male !!
3 BB87.6          male !!
4 BB90.3          female*
5 SS91.3           female !!
6 SS91.4           female !!
7 SS91.5           male
8 SS93.3           female
9 SS93.4           male !!
10 SS93.5         male !!
11 Pos (male)
12 Neg (female)
13 Neg (dH2O)
14 Ladder    








!! these are the correct gender that I needed. I'll probably use BB87.6M and SS91.4F (and not SS932F and SS93.4M). Finally to round out the P. campbelli I will use BB77.3M.

*this sample seemed very degraded, won't use for RNAseq regardless, but it may be "female" due to failure of the PCR (rather than no Y chromosome) as there was very little DNA in the extraction...


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Auer, P. L. and R. W. Doerge. 2010. Statistical Design and Analysis of RNA Sequencing Data. Genetics 185:405–416.

DeVeale, B., D. van der Kooy and T. Babak. 2012. Critical Evaluation of Imprinted Gene Expression by RNA–Seq: A New Perspective. PLoS Genet 8:e1002600.

Gregg, C., J. Zhang, B. Weissbourd, S. Luo, G. P. Schroth, D. Haig and C. Dulac. 2010. High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Mouse Brain. Science 329:643–648.

Gregg, C., J. Zhang, J. E. Butler, D. Haig and C. Dulac. 2010. Sex-Specific Parent-of-Origin Allelic Expression in the Mouse Brain. Science 329:682–685.

Proudhon, C. and D. Bourc'his. 2011. Identification and resolution of artifacts in the interpretation of imprinted gene expression. Briefings in Functional Genomics 9:374–384.