3700 sequencing problems

Hall, L.S. lsh11 at leicester.ac.uk
Fri Dec 19 06:13:09 EST 2003

	 -----Original Message-----
	From: 	Hall, L.S.
	Sent:	15 December 2003 13:00
	To:	'ABRF Discussion List'
	Subject:	3700 sequences

	Dear Colleague(s)

	I am currently faced with what should be an 'easy fix' problem but in reality has been dragging on for months: Specifically, we possess a 3700 and of late, samples that sequenced very well have started to fail or produce top heavy sequence with alot of noise; What is even more curious is the fact that all samples produce better sequence in terms of balanced signal and superior signal to noise if the same samples on the same plate are rerun. Nevertheless, in nearly all cases even those sequences that second time round produce raw data demonstrating an optimal signal strength and a good spread in terms of 5' -3' signal demonstrate background contamination, invariably amorphous red 'noise' under sharp blue specific peaks. On face value and to my mind this might suggest the following:

	1. The consistent/persisitent presence of one color under another may be a matrix artifact, necessitating a new spectral assay.

	2. The top heavy peaks and overall weak data might be due to some sort of salt contamination in the sequencing preparations; Further, augmented signal from reruns of the same sample could be due to any salt present being depleted or eradicated during the initial injection. Consequently, when the same 'desalted' sample is reinjected by rerunning the same plate, extension products in general and 3' products in particular are more efficiently injected because salt is absent or considerable reduced.

	Considering the above, I have made multiple preps of my templates ( PCR) using different preparation procedures and changed all of my reagents including primers and HiDi formamide ( which incidentally I have started to desalt as well using mixed bed ion exchange resins) in an effort to minimise putative salt contamination but poor data persists ?? Further, as a control for template preparations, I have sequenced commercial preparations of pGEM which, in the past, have yielded expected excellent sequence but at the moment are demonstarting poor quality sequence by virtue of being top heavy, of low signal strength and contaminated with this 'red under blue artifact, regardless of signal strength ?? With regard to poor spectral correction, the red under blue affect persists in spite of running new spectrals and verifying the quality of resulting assay for individual capillaries ( which in terms of visual data and QC parameters like condition number and 'Q' value are apparently fine ??)

	I will add one more thing for now: In view of such problems and times scales involved, AB have looked at the machine a number of times, identified and fixed obvious defects, e.g. a loading artifact; defective cuvette, but the problem was still manifest with last plate of PGEM's I ran ?? The other thing of note which one would think vindicates the 3700 itself is the fact that the long read standards consistently demonstrate excellent extracted data although, nonetheless, raw signal strength does increase from one run to the next ( which might imply that unless solute contamination is coming from the Hi Di itself the problem might reflect defective electro kinetic injection ??).

	I have been sequencing pGEMs inter alia for over 7 years and have never experienced anything quite like this ?? At this juncture considering the above I am truly baffled. Doeas anyone have any explanation as to what may be involved or things that I might try next to clarify the nature of the problem (s) ??

	Thanks for any help,

	Laurence Hall

Laurence Hall
Cardiovascular Sciences
University of Leicester
Glenfield Hospital
Leicester LE3 9QP

Tel.: 0116 256 3040
Fax:  0116 287 5792
E-mail: LSH11 at le.ac.uk


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