Genome Centre

SNP Genotyping Service

The SNP (single nucleotide polymorphism) Genotyping service uses a state-of-the-art Sequenom MALDI-TOF mass spectrometry based system (see above), which is capable of high-throughput genotyping (>10 000 individual genotypes per day).

Essentially, the service consists of the following steps:

1. SNP choice by user
2. SNP assay design by service or user
3. Primers ordered by user
4. PCR carried out by user
5. Genotyping carried out by service and results sent to user

New users are advised to discuss project design beforehand with the Genome Centre manager, Dr Andrew Walley, as poor study design could be costly.

Detailed breakdown of stages:

1. SNP CHOICE is down to the user but a few points should be noted.
   • Initially only simple base changes will be typed, i.e. not insertion/deletions or repeat length variation
   • SNPs close together (<40bases) can usually be typed but should be discussed prior to submission
   • SNP resources include NCBI dbSNP, The SNP Consortium, the HUGO Mutation Database list, the EMBL Mutation database list and SNPper. Whole genome sequences are available using the University of California at Santa Cruz genome browser.
2. SNP ASSAY DESIGN is provided as a service to users. A minimum of 50 bases of sequence either side of the SNP should be provided, ideally 200 bases either side. The sequence should be in upper case and the SNP should be designated by square brackets, e.g. AGCC[G/A]GTTC. Use the IUB codes for ambiguous bases if there are unknown or other polymorphic bases in the sequence, e.g. “N” for unknown or “Y” for C or T. The data should be organised in a tab-delimited text format as a two-column table (e.g. create in Excel and save as a text file). The headings of the columns should be SNP_name and SNP_sequence respectively. Each SNP name must at least be unique in the file and users MUST restrict character use to standard letters and numbers, space ( ), hyphen (-) and underscore (_) only. Non-standard files will be returned to users. If users have information on problematic sequence, e.g. Alu repeats, then this can be highlighted by using lower case. The SNP text file should currently be emailed to Andrew Walley . Output files will be emailed back.
   N.B.For users with access to the Sequenom design software they can carry this stage out themselves. PCR products should be in the range of 80-120 bp. All extension products should differ by at least 50Da and the range of extension product sizes should be 5000-8500Da (approx. 15-25 bases). Multiplexes of 3-5 are recommended, users wishing to go higher should discuss this first.
3. PRIMERS should then be ordered by users based upon the output of the Sequenom primer design software. In the file there are three primer sequences: the two PCR primers (2nd-PCRP and 1st-PCRP) and the extension primer (UEP_SEQ) in columns D, E and N respectively. The only Sequenom approved oligonucleotide manufacturer is Metabion and the PCR primers should be ordered at 0.04OD scale and the extension primer at 0.2OD scale from them. Both PCR and extension primers have been ordered from Sigma-Genosys and there have been no obvious problems.
4. PCR REACTIONSare to be performed by individual users. Access to the Genome Centre’s 384-well MJR PCR tetrads will be on a first-come first-served basis, provided that the Sequenom service does not need them. A booking form will be provided adjacent to the machines for bookings, together with rules of use.

All PCR reactions should be performed in an ABgene 384 well PCR plate. Reaction volume total is 5µl. All plates should be sealed with ABgene adhesive PCR film.

PCR reaction mix (4x multiplex):

	x1 (in µl)	x 460 (in µl)
dH2O	2.18*	1002.8
Qiagen Hot Star 10X Buffer	0.50	230
Qiagen Hot Star MgCl2 (25mM)	0.20	92
dNTP (10mM)	0.1	46
Primer Mix (1µM) **	0.5	115
Qiagen HotStarTaq (5U/µl)	0.02	9.2
DNA (2.5ng/µl)	1.5	1.5

*Adjust amount of water according to number of primers used

**per primer pair in the multiplex reaction, e.g. 4-plex will be total of 8 primers x 0.25µl = 2 µl. The initial primer pair mix needs to be more concentrated for multiplexes higher than 6x.

Do not use any other reagents in your PCR as they are very likely to adversely affect the matrix on the SpectroCHIP.

PCR cycling conditions:

Temperature	Time	Cycles
95°C	15min	1
95°C	20s	4
65°C	30s
72°C	1min
95°C	20s	4
58°C	30s
72°C	1min
95°C	20s	38
53°C	30s
72°C	1min
72°C	3min	1

N.B. These conditions routinely perform significantly better than those recommended by Sequenom Inc.

The following must accompany the plate to be genotyped:

1. A completedRequest form – containing plate name, SNP Assay details and locations, termination mix to be used and the name(s) of the sample list file(s).

       2. Extension primers diluted to 100µM

       3. The Primer Design spreadsheet containing the assay information.

       4. A sample sheet listing sample locations on the plate. Fill in your sample list vertically in column A of an MS-Excel spreadsheet in the order A1 = well A1, A2 = well B1, A3 = well C1, etc., i.e. reading vertically down consecutive columns. Save as a tab-delimited text file (default .txt file extension). If unsure, open it in a text editor like Windows Notepad to ensure it is plain text.

The three files (order form, primer design file and sample sheet list) need to be emailed to Marlene Attard m.attard@imperial.ac.uk

The plates are to be placed in the Sequenom freezer in the main Genome Centre laboratory. The details of the plate submitted to the queue should be entered into the queue folder kept on the freezer.

On completion of genotyping, the data will be emailed to the user as a tab-delimited text file suitable for opening in MS-Excel.

 

Costs

Please contact Marlene Attard for details of costs.