3730 genetic analyzer

Patient blood DNA was extracted using Promega Maxwell 16 Blood DNA Purification Kits (Promega). Primers were designed adjacent to the estimated deleted region boundaries, and a Qiagen Taq PCR Core Kit was used to amplify the deletion boundaries. DNA fragments were gel‐purified using E‐Gel SizeSelect Gels (Life Technologies). DNA sequencing was performed by PCR using a Qiagen Taq PCR Core Kit (Qiagen) according to the manufacturer's specifications, followed by bidirectional sequencing using the Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's specifications and run on an ABI 3130xl or 3730 Genetic Analyzer (Applied Biosystems). Sanger sequencing was conducted at the CCR Genomics Core at the National Cancer Institute, NIH, Bethesda, MD. Forward and reverse sequences were evaluated using Sequencher 5.0.1 (Genecodes). All deletion breakpoint coordinates and Alu locations are based on the GRCh37/hg19 genome build.

HLA class I genotypes were determined by either the PCR–sequence-based typing protocol, as recommended by the 13th International Histocompatibility Workshop or by targeted next-generation sequencing, as described (11 (link)).
DNA from individuals in the three South African cohorts and the NCI RDP (the majority of the NCI RDP are of European descent) was sequenced for the 5′ UTR, exons, and 3′ UTR of TAPBP. Three gene segments were amplified with Platinum Taq DNA polymerase (Invitrogen) in several fragments: from the 5′ UTR to the beginning of the third intron (approx. 1.5 kb), from the end of the third intron to the beginning of the seventh intron (approx. 1.5 kb), and from the end of the seventh intron to the end of the 3′ UTR (approx. 2.1 kb) (see SI Appendix, Table S7, for primer sequences). The amplified fragments were sequenced by using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and run on a 3730 Genetic Analyzer (Applied Biosystems) (primers used for sequencing are listed in the SI Appendix, Table S7). rs111686073 and rs59097151 were genotyped in the individuals from the Ugandan cohorts by amplifying the two regions containing the SNPs with Platinum Taq DNA polymerase (Invitrogen) (primers used for amplification and sequencing are listed in the SI Appendix, Table S7).

The PCR primers were designed using the online MethPrimer software (http://www.urogene.org/methprimer/index.html). Genomic DNA extractions of IUGR/NBW 5–10 were performed using the same method as mentioned above. 400 ng of DNA samples were converted using ZYMO EZ DNA Methylation-Gold Kit^™ (ZYMO) and one-third of the elution products were used as templates. PCR amplification was carried out with a thermal cycling program of 94 °C for 1 min, 30 cycles of 94 °C for 10 s, 58 °C for 30 s, 72 °C for 30 s, and then final 5 min incubation at 72 °C. PCR products were purified using the QIAquick Gel Extraction Kit (QIAGEN) and subcloned. Twenty-four colonies from each product were sequenced using the 3730 Genetic Analyzer (Applied Biosystems). Then, the reads were post-processed and aligned to the pig reference regions (all PCR regions) using SOAP aligner (Version 2.01) after sequencing according to a previously published method with default parameters that excluded reads with more than five mismatched bases. Multiple reads mapping to the same position were counted only once to remove potential bias from PCR.

Genomic DNA was extracted from whole blood with a QiAmp® DNA blood purification kit (Qiagen). BRCA1 and BRCA2 exons were amplified by PCR with specific primers located in intron/exon boundaries. Twenty-eight fragments covering the 22 coding exons of BRCA1 and 32 fragments covering the 26 coding exons of BRCA2 gene were amplified.^{48 (link)} The large exons 10 and 11 of BRCA2 were amplified as two and nine fragments, respectively, while exon 11 of BRCA1 was amplified as seven fragments (Supplementary Table 1). PCRs were carried out with initial denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a GeneAmp® PCR System 9700 (Applied Biosystems) as described previously.^{48 (link)}The PCR products were purified with a MinElute 96UF kit and sequenced using a Big Dye terminator V3.1 sequencing kit on a 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Both forward and reverse strands were sequenced. Obtained sequences were compared with BRCA1 GenBank reference sequence (NM_007294.3) with Alamut Software. This method was used for the first 15 index cases recruited.

Genomic DNA was isolated from peripheral blood samples using the Blood Genomic DNA Miniprep Kit (Axygen, CA, USA). MLPA P245 kits (MRC-Holland, Amsterdam, the Netherlands) were used to screen common microdeletion syndromes following the manufacturer's instructions. Briefly, DNA was denatured at 98 °C for 5 min and hybridized with the probes at 60 °C for 16 h. Ligation was performed at 54 °C for 15 min, and ligated probes were subsequently amplified by PCR using universal fluorescent primers. The fragments were separated by capillary electrophoresis using the 3730 Genetic Analyzer (Applied Biosystems, CA, USA) and analyzed using Coffalyser software (MRC-Holland).