Any experience with Paired-end on XL+ (upgraded 454)?

[Indiana]

We did our first PE run since the XL+ upgrade. I'm told PE runs are "not supported" on the platform. The results were high keypass, but very low filter pass (see below and attached). Images were VERY bright near the beginning of the run. Should I try reducing the amplification primer concentration? Seems like PE libraries had 1/4 the amount of primer in the previous FLX-Titanium protocol.

TCAG (Library) Region ----- 1 ----- 2 -----Total
Raw Wells -----1,130,136----- 1,122,335------ 2,252,471
Key Pass Wells -----1,082,488------ 1,092,787 ------ 2,175,275
Passed Filter Wells----- 122,314----- 129,766 ----- 252,080
Total Bases ----- 30,312,331 ----- 31,471,974 -----61,784,305

Length Average ------- 247.82------ 242.53 ----- 245.1
Length Std Deviation ----- 170.65 ----- 162.18
Longest Reads Length -----1,596 -----1,596 -----1,596
Shortest Reads Length -----40----- 40 -----40
Median Reads Length -----216 -----217 -----216

Dot -----45,115 ----- 47,739 ----- 92,854
Mixed -----329,041----- 304,870----- 633,911
Short Quality -----531,666 -----579,124 -----1,110,790
Short Primer -----4----- 6 -----10

% Dot + Mixed ----- 34.56----- 32.27----- 33.41
% Short ------- 49.12----- 53 ----- 51.06
% Passed Filter ----- 11.3----- 11.87 -----11.59

All ControlDNA Wells----- 5,765 -----5,145 -----10,910
Average
600 bp, 100% ----- 0.75 ----- 1.26 ----- 0.99
>=98%----- 17.71 -----19.65 -----18.63
>=95% -----32.33 -----34.58 -----33.39

[pmiguel]

Hi Indiana,
Did you do a bioanalyzer chip on the library prior to emPCR?
We don't have the FLX+ upgrade yet. But that sounds like short (100-200 bp) library molecules in your library.

--
Phillip

[MissDNA]

What was the length avg for the controls? If they are shorter than usual then you might have had some equipment or reagent problem, if not then it might have been your library prep. As pmiguel said there is a lot of short fragments.

I have only run 3kb PE libraries using the regular titanium chemistry and avg size is always around 350-380 bp.

Our upgrade will finally happen on the 3rd. I do not intend to use FLX+ kits for PE preps. Btw did you run 200 or 400 cycles?

[RCJK]

What emPCR conditions did you use? Those for the XLR70 kit (Ti emPCR) or those for the XL+ kit (XL+ emPCR)? I wonder if some of what happened is that the software is expecting longer fragments and isn't happy about the shorter length fragments that were actually sequenced. I'm sure they have a reason as to why they do not support the PE libraries or amplicon libraries on the XL+ kits, it would just be nice if they shared that info.