| | The following method definition consists of one or more |
| | protocols used in The SNP Consortium project by the |
| | participating laboratory SANGER. |
| | See the TSC website (http://snp.cshl.org/cgi-bin/protocol) |
| | for a complete list of TSC protocols. |
| | \\--------------------------------------- |
| | SNP production protocol TSCM0019 ( |
| | http://snp.cshl.org/cgi-bin/protocol?id=TSCM0019) |
| | Title: Library preparation and sequencing for PvuII enzyme |
| | Lab: Sanger Center, Cambridge |
| | Contact: [email protected] |
| | Description: |
| | A panel of 24 DNAs was digested with PvuII, size |
| | fractionated on an agarose gel, and cloned into puc18-based vectors. |
| | Size fractions were taken at 1.0-1.1kb, 1.1-1.2kb, 1.2-1.4kb, 1.4-1.6kb, |
| | 1.6-1.8kb, 1.8-2.1kb, 2.1-2.5kb, 2.5-3.0kb, 3.0-3.5kb and 3.5-4.0kb, |
| | from which libraries were constructed. Samples from these libraries were |
| | named such that the size insert can be identified. The sample names |
| | start with p1_0, p1_1, p1_3, p1_5, p1_7, p1_9, p2_3, p2_7, p3_2 and |
| | p3_7, with respect to the size fractions listed above. Sequences were |
| | obtained primarily from ABI 3700 capillilary sequencers. Base-calling |
| | was performed with Phrap, which provides the quality values upon which |
| | the SNP detection is based. |
| | \\--------------------------------------- |
| | SNP detection protocol TSCM0026 ( |
| | http://snp.cshl.org/cgi-bin/protocol?id=TSCM0026) |
| | Title: SNPs from reads detected using SsahaSNP from public human genomic sequence |
| | Lab: Sanger Center, Cambridge |
| | Contact: [email protected] |
| | Description: |
| | All reads were clipped of sequencing vector and low quality ends, |
| | which set a usable read length for each read. The clipped reads |
| | were screened for repetitive sequence with RepeatMasker, using |
| | the default human settings. Only reads with >=80 non-repetitive |
| | bases and >= 100 Phred quality (Q) >=30 bases were used in this |
| | analysis. The reads were aligned using SsahaSNP to available |
| | finished and unfinished (with quality values) public human |
| | genomic sequence which is available via anonymous ftp at |
| | snp.cshl.org in the files pub/SNP/gbfinished* and pub/SNP/nr*. |
| | All reads which aligned to genomic sequence within 50bp of the |
| | start and 60bp of the end of their usable read length, were as- |
| | sembled with the matching region of genomic sequence (Q was set |
| | to 40 for finished genomic sequence). High quality base |
| | discrepancies (Q>=23) were identified as candidate SNPs. Further |
| | restrictions on the candidate SNPs were that its neighbouring 5 |
| | bases all had Phred quality values of >=15 and at least 9 of the |
| | 10 neighbours match. If the number of detected SNPs in one clique |
| | was greater than 4 then all SNPs were discarded for that |
| | clique. |
