Sample preparation
An amplicon library of 34 individuals was prepared by PCR of the Dog Leukocyte Antigen gene DLA-DRB1 exon 2 (target size: 303 bp) [13], introducing identifying tags and the common Roche/454 A/B FLX sequences. The individuals had previously been shown to be heterozygous for this gene by traditional Sanger sequencing. The library was analyzed by agarose gel electrophoresis and ethidium bromide staining. Approximately 400-bp long fragments were extracted by gel purification and the product was then analysed by agarose gel electrophoresis and GelRed (Biotium, Inc., Hayward, CA, USA) staining. EmPCR and strand-separation were carried out according to instructions of the manufacturer of the sequencing system (Roche). Briefly, an emulsion was formed by shaking "mock buffer" with mineral oil, then PCR reagents, the DNA library and beads were shaken into the emulsion. Following clonal amplification of library fragments on beads, the emulsion was broken and beads were collected by centrifugation. For labelling and subsequent FACS enrichment, the protruding DNA strands were removed by washing the collected beads in 0.1 M NaOH according to the manufacturer's protocol. For comparison, a control emPCR reaction mixture was prepared from the same library, and unspecific enrichment of DNA-covered beads was carried out, using streptavidin-covered beads, again according to the manufacturer's instructions.
emPCR product labelling
For detecting beads carrying the amplified gene fragment by FACS, a biotinylated 50 mer was designed to match the central part of the amplicon product and still allow for some mismatches in the hypervariable DLA target sequence. To investigate whether the amount of DNA on the beads could be visualised by FACS, beads were also labelled with random hexamers and SYBR Green.
Dual labelling was carried out by adding 20 pmol random hexamers and 20 pmol biotinylated gene-specific 50 mer probe, both from Eurofins MWG Operon (Ebersberg, Germany) in a total volume of 100 μl 1× Annealing Buffer (1×AB, Roche, Basel, Switzerland) and applying the following conditions: 1 min at 95°C, then decreasing to 25°C over 10 min. 1 μl 50 × SYBR Green I Nucleic Acid Gel Stain (SYBR Green, Molecular Probes Inc., Eugene, OR, USA) and 4 μl streptavidin-conjugated Alexa647 dye (0.2 mg/ml, Invitrogen, Carlsbad, CA, USA) were then added to each sample, and the resulting mixture was incubated for 10 min at room temperature. Samples were then transferred to 5 ml Polystyrene Round-Bottom FACS Tubes (BD Biosciences, Franklin Lakes, NJ, USA) and diluted by adding 500 μl phosphate-buffered saline (PBS) containing 0.1% Pluronic F108 NF surfactant (PBSP) (BASF Corporation, Mount Olive, NJ, USA). As negative FACS control, unreacted beads were labelled in the same manner.
FACS enrichment
FACS enrichment of beads carrying an amplified gene fragment on their surface was achieved by sorting out beads that were Alexa647 positive using a FACSAria instrument (Beckton Dickinson, San Jose, California). Beads were transferred to Ultrafree®-MC Centrifugal Filter Units (Millipore, Billerica, MA, 0.65 mm pore size) and washed twice in 140 μl 0.1 M NaOH for 40 s and twice in 250 μl 95°C 1×AB for 40 s, followed by re-suspension in 1×AB.
Sequencing
FACS-enriched and standard-enriched control samples were sequenced in separate 1/16-lanes of a picotiter plate according to the instructions of the sequencing system.
Sequence analysis
Sequences were sorted by individual using an in-house developed Perl script, aligned using Muscle [14], Jalview version 2 [15] was employed for visualisation and analysis of the alignments.