BA Traces what's going on?

[subxero]

Quick summary,

some library prepped using truseq RNA v2 kit slightly modified protocol (mainly volumes)

Samples 1-8 are library I prepped last week (my first time), some samples at different RNA input conc. (RNA from human blood) PAX gene, and globin cleared (some samples Double globin cleared)
samples 9-11 library prepped few weeks ago by experienced tech using same protocol.
BA was done with a DNA high sensitivity chip

can anyone tell me what they think is going on?

Why are my peaks so big?
Why do a few have a double main peak?
What is the broad trailing peak on some of the samples?
In samples 9-11 I assume early peak is dimers, that is not good right? Dimers will use up some of my reads right?

[kwaraska]

The "double peak" is likely too much DNA. It goes off scale and that is what you see.

Yes-the first peak in the later traces are likely primer dimer-they will take up sequencing real estate.

[microgirl123]

Those aren't pretty! The double peak may be offscale. The secondary peak to the right may be from overamplification (was there a PCR step after you made your library?). The peak @140 bp is adapter dimer. If you don't get rid of that you'll waste a lot of space on your flow cell as adapter dimer sequences preferentially.

[subxero]

Yeah there was a PCR step to amplify the Library, I figured I had over amplification but still don't understand how my samples are over amplified yet the samples prepped a few weeks prior using all the same stuff does not?

What is common cause of adapter dimers?

[microgirl123]

Adapter dimers can result when your DNA:adapter ratio is off, among other things. If you have them and then do PCR, they amplify rapidly and happily. I generally use an 0.6x concentration of Ampure XP beads to remove them - it's not always perfect, but it usually gets rid of most of them.

[subxero]

Ill definitely look into another bead clean up step.

how much total library should I be aiming for in the end?

[microgirl123]

What instrument are you planning on running your libraries on?

[subxero]

cluster with cbot then run on HiSeq2000

I guess the big question is, what do I have to do to get this library to a point where it can be sequenced efficiently?

A) get ride of dimers
B) decrease amplification (simply decrease PCR cycles?)

But other than that it looks reasonable?

[subxero]

Here is a trace from my latest library prep. I ran these libraries on a DNA 1000 chip instead of the HS chip as I figured had enough library to do so.

I still need to do the kappa qPCR QC to get my concentrations but to my untrained eye things look good? reasonable? somewhere in between maybe.

here are values to go with trace

Size [bp] Conc. [ng/µl] Molarity [nmol/l] Observations
15 4.2 424.2 Lower Marker
253 2.75 16.5
258 0.63 3.7
265 0.71 4
276 0.62 3.4
286 2.25 11.9
310 0.82 4
320 0.67 3.2
331 1.28 5.8
356 0.37 1.6
365 0.28 1.2
372 0.24 1
379 0.26 1.1
1,500 2.1 2.1 Upper Marker