Wrong library size reads

[soleulloa]

Hi all,

I've prepared a library using the protocol for 200 bp reads and checked by Bioanalyzer library size was 238 bp.
Then, OT runs and PGM runs, but the readings obtained were too short (53 bp mean, 42 bp median and 28 bp mode).
What happened?
Maybe the emulsion was broken? Why the emulsion could be broken?

I hope your answers!!!

Thank you and have a nice day

Soledad

[Sharp_GeCKO]

How long is your target sequence？
I guess that your total length of A adaptor+barcode+target fragment is nearly 53bp. So the rest 238-53=185bp might be the same sequence. When PGM runs, it can not recognize and correct the bases if all the cores on the chip are the same bases. It is just like that all the lights shining at the same time, then you can not see anything.

To solve this problem, you should redesign your primer by adding 1-9bp variable length sequence between your A adaptor and barcode.

[snetmcom]

How long is your target sequence？
I guess that your total length of A adaptor+barcode+target fragment is nearly 53bp. So the rest 238-53=185bp might be the same sequence. When PGM runs, it can not recognize and correct the bases if all the cores on the chip are the same bases. It is just like that all the lights shining at the same time, then you can not see anything.

To solve this problem, you should redesign your primer by adding 1-9bp variable length sequence between your A adaptor and barcode.

This is incorrect. I sequence fragments with homologous bases for the first 30+ bases all the time.

It sounds more like poor quality sequencing or an issue with the library.

[MrGuy]

How did you do your size selection?
How did you quantify you library prior to OT2?
Are all your sequencing reagents fresh and from the same package?