GBS sequencing of the Oregon Wolf Barley population
Oligonucleotides comprising the top and bottom strands of each barcode adapter and a common adapter were diluted (separately) in TE (50µM each) and annealed in a thermocycler (95°C, 2 min; ramp down to 25°C by 0.1°C/s; 25°C, 30 min; 4°C hold).  Adapters were then quantified using an intercalating dye (picogreen; Invitrogen, Carlsbad, CA), diluted in water to 0.6 ng/µL, and 6 µL (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 µL) were added to individual adapter-containing wells and plates were, again, dried.    Samples (DNA plus adapters) were digested for 2 h at 75°C with ApeKI (New England Biolabs) in 20 µL volumes containing 1X NEB Buffer 3 and 3.6U ApeKI .  Adapters were then ligated to sticky ends by adding 30 µL 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 22°C for 1 h and heated to 65°C for 30 min to inactivate theT4 ligase.  Sets of 48 or 96 digested DNA samples, each with a different barcode adapter,  were combined (5µL 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 50µL.   Restriction fragments from each library were then amplified in 50µL volumes containing 2 µL 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 72°C for 5 min, 98°C for 30 s followed by 18 cycles of 98°C for 30 s, 65°C for 30 s, 72°C for 30 s with a final Taq extension step at 72°C for 5 min.   These amplified sample pools constitute a sequencing “library”.   Libraries were purified as above (except that the final elution volume is 30 µL) and 1 µL 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.  The bar code of each sample in the multiplexing libraries is attached below. Each sequence read should start with <barcode>C[A or T]GC.    Lane barcode sample  7 CTCC OWB003  7 TGCA OWB018  7 ACTA OWB038  7 GTCT OWB055  7 GAAT OWB072  7 GCGT OWB090  7 TGGC OWB010  7 CGAT OWB030  7 CTTGA OWB047  7 TCACC OWB060  7 CTAGC OWB081  7 ACAAA OWBDom  7 TTCTC OWB006  7 AGCCC OWB020  7 GTATT OWB041  7 CTGTA OWB056  7 AGCAT OWB075  7 ACTAT OWB091  7 GAGAAT OWB013  7 CCAGCT OWB031  7 TTCAGA OWB051  7 TAGGAA OWB064  7 GCTCTA OWB082  7 CCACAA OWBRec  7 CGTCAA OWB007  7 CTTCCA OWB023  7 GAGATA OWB042  7 ATGCCT OWB057  7 TATTTTT OWB076  7 CTTGCTT OWB092  7 ATGAAAC OWB014  7 AAAAGTT OWB033  7 GAATTCA OWB052  7 GAACTTC OWB068  7 GGACCTA OWB084  7 GTCGATT OWBDom  7 AACGCCT OWB009  7 AATATGC OWB028  7 ACGACTAC OWB043  7 AAGGATGC OWB059  7 TAGCATGC OWB077  7 TAGGAAGC OWB093  7 TAGGCCAT OWB016  7 TGCAAGGA OWB034  7 TGGTACGT OWB053  7 TCTCAGTC OWB069  7 CCGGATAT OWB087  7 CGCCTTAT OWBRec