title: GBS sequencing of NAM plate 705VVAAXX_7
design: Oligonucleotides comprising the top and bottom strands of each barcode adapter and a common adapter were diluted (separately) in TE (50 microM each) and annealed in a thermocycler (95C, 2 min; ramp down to 25C by 0.1C/s; 25C, 30 min; 4C hold). Adapters were then quantified using an intercalating dye (picogreen; Invitrogen, Carlsbad, CA), diluted in water to 0.6 ng/microL, and 6 microL (3.6 ng) of each pair of adapters (barcode and common) was aliquoted into a 96-well PCR plate and dried down. DNA samples (100ng in a volume of 10 microL) were added to individual adapter-containing wells and plates were, again, dried.    Samples (DNA plus adapters) were digested for 2 h at 75C with the restriction enzyme specified in the sample description. Adapters were then ligated to sticky ends by adding 30 microL of a solution containing ligase buffer with ATP (1.66 X) and T4 ligase (640 cohesive end units) (New England Biolabs) to each well.  Samples were incubated at 22C for 1 h and heated to 65C for 30 min to inactivate theT4 ligase.  Sets of 48 or 96 digested DNA samples, each with a different barcode adapter,  were combined (5 microL each) and purified using a commercial kit (QIAquick PCR Purification Kit, Qiagen) according to the manufacturer's instructions. DNA samples were eluted in a final volume of 50 microL.   Restriction fragments from each library were then amplified in 50 microL volumes containing 2 microL pooled DNA fragments, 1X Taq Master Mix (New England Biolabs) and 25 pmol, each, of the following primers: (A) 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT and (B) 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT.   These primers contained complementary sequences both for amplifying restriction fragments with ligated adapters, and for facilitating downstream sequencing (i.e., binding PCR products to oligonucleotides that coat the Illumina sequencing flow cell and priming DNA sequencing reactions).     Temperature cycling consisted of 72C for 5 min, 98C for 30 s followed by 18 cycles of 98?C for 30 s, 65C for 30 s, 72C for 30 s with a final Taq extension step at 72C for 5 min.   These amplified sample pools constitute a sequencing library. Libraries were purified as above (except that the final elution volume is 30 microL) and 1 microL was loaded onto an Experion automated electrophoresis station (BioRad) for evaluation of fragment sizes.   Libraries were considered suitable for sequencing if adapter dimers (~128 bp in length) were minimal or absent and the majority of other DNA fragments were between 170-350 bp. If adapter dimers were present in excess of 0.5% based on the Experion output, libraries were constructed again using a few DNA samples and decreasing adapter amounts.  Once the appropriate quantity of adapters was empirically determined for a particular enzyme/species combination, no further adapter titration was necessary.  Single-end sequencing (86 bp reads), one 48- or 96-plex library per flowcell channel, was performed on a Genome Analyzer II (Illumina, Inc.).   See Bentley et al (2008) for details of the sequencing process and chemistry.