Insert length for RNAseq

For this big sequencing thing, I'm planning on using a 100bp PE protocol. This means that the Illumina machine will read the sequence from both ends and give me a sequence from each. The main question I'm working on now is how long should the insert size be? 

If it is less than 200bp then I will get overlapping sequences (which is good according to J~, although I'm not clear as to exactly why). However Sara just got some sequencing back which had (we think) too much overlap and this diminished the total amount of data she has by about 10%. Her insert size was ~150bp which means the overlap was 50%. I think that what I should shoot for is closer to 20% but I can't seem to find any papers or seq-answer threads that address the issue.

I think that the reason to have some overlap is that the program I'll be using, Trinity (Grabherr 2011), will function better, but I'm not really sure. 

Hopefully I can get this figured out soon so that I can start preparing my libraries. I have all the samples extracted so that's the final step before they get sent off. 

3-10-13 update: The TruSeq protocol uses a enzyme to fragment the mRNA and then a bead-based size selection. I can modify this size-selection, but at this point it seems prudent to just go ahead and use what they give me.

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Grabherr, M. G., B. J. Haas, M. Yassour, J. Z. Levin, D. A. Thompson, I. Amit, X. Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A. Gnirke, N. Rhind, F. Di Palma, B. W. Birren, C. Nusbaum, K. Lindblad-Toh, N. Friedman and A. Regev. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology 29:644–652.

RNAseq Samples

Here is the running list of my samples for RNAseq, their RIN numbers, concentration, and library prep. I will keep updating this as I go until I have all 40 samples extracted and the libraries prepared.

#	Date extracted	ID	RIN	[ ] ng/uL	Library
1	9-28-12	BB.BB15.3M	9.6	1071	Yes
2	9-28-12	BB.SS70.3M	9.6	1127	Yes
3	9-28-12	SS.BB20.2M	9.7	859	Yes
4	9-28-12	SS.SS82.1M	9.8	1543	Yes
5	9-28-12	BB.BB15.4F	9.4	1690	Yes
6	9-28-12	BB.SS70.2F	9.4	1427	Yes
7	9-28-12	SS.BB20.6F	9.6	1818	Yes
8	9-28-12	SS.SS82.2F	9.6	1808	Yes
9	9-29-12	BB.BB77.2M	9.3	3086	Yes
10	9-29-12	BB.SS70.5M	9.4	5631	Yes
11	9-29-12	SS.BB25.5M	9.7	1783	Yes
12	9-29-12	SS.SS87.1M	9.5	2363	Yes
13	12-15-12	BB.BB77.1F	9.4	512	Yes
14	12-15-12	BB.SS70.4F	9.3	367	Yes
15	12-15-12	SS.BB25.3F	9.4	553	Yes
16	12-15-12	SS.SS87.3F	9.6	455	Yes
17	12-15-12	BB.BB86.2M	9.4	449	Yes
18	12-15-12	BB.SS72.2M	9.6	483	Yes
19	12-15-12	SS.BB25.3M	9.3	326	Yes
20	12-15-12	SS.SS88.1M	9.6	440	Yes
21	12-15-12	BB.BB86.1F	9.1	235	Yes
22	12-15-12	BB.SS72.1F	8.9	354	Yes
23	12-15-12	SS.BB29.1F	9.1	266	Yes
24	12-15-12	SS.SS88.3F	9.5	507	Yes
25	12-17-12	BB.BB87.6M	9.1	466	Yes
26	12-17-12	BB.SS73.3M	8.6*	466	Yes
27	12-17-12	SS.BB20.8M	9.0	354	Yes
28	12-17-12	SS.SS89.2M	9.0	254	Yes
29	12-17-12	BB.BB87.1F	9.0	411	Yes
30	12-17-12	BB.SS73.1F	9.1	414	Yes
31	12-17-12	SS.BB24.2F	8.7	257	Yes
32	12-17-12	SS.SS89.1F	9.0	394	Yes
33	2-19-13	BB.BB77.3M	9.1	519	Yes
34	2-19-13	BB.SS71.3M	8.4	284	Yes
35	2-19-13	SS.BB22.4	8.5	273	Yes
36	2-19-13	SS.SS91.1M	9.5	557	Yes
37	2-19-13	BB.BB90.1F	8.3	448	Yes
38	2-19-13	BB.SS73.2F	8.4	318	Yes
39	2-19-13	SS.BB24.3F	8.1	281	Yes
40	2-19-13	SS.SS91.4F	8.5	563	Yes

12-17-12: I was going to try to finish everything up today or tomorrow, but I ran out of reagents. I ordered more but they won't get in for a little while. I'm really frustrated as I was hoping to finish everything up before I head home for the break. Now it looks like it won't get finished until February.

12-18-12: It's good that I didn't extract the other 8 yesterday, as the RIN numbers were all quite low. I'm not sure why, but I need to get that straightened out. I got one of the reagents in the mail today, now I'm just waiting on the other and I will be set to finish everything off.

2-20-13: Just bioanalyzed the last samples. Not great, but D~ says they are good enough - Illumina calls for >8. J~ was fine with it since there are only 8/40 that are between 8 and 9.

*There is one sample that was only 7.4 (BB.SS73.3M) and I re-bioanalyzed it today it went down to 5.6. This says to me that it has some RNase contaminant in it and is being actively degraded. I have some RNA partially extracted and archived that I will finish extracting, but most likely I will need to find a new sample. Though, having 1 out of 40 go bad is fine by me, that's pretty good odds.
*4-1-13 Upon re-extracting this sample the RIN was 8.6 and the concentration was a bit high (so the actual RIN is likely much higher) I can now finish up with the library preps.