Wheat exome capture and targeted resequencing
Illumina Paired End library preparation   Fragmentation  5ug of pooled gDNA was fragmented using a UCD-200 biorupter (Diagenode) on high setting (30s on/30s off) for 35min to get an average fragment size of 250bp.  Fragmented samples were purified down a Qiagen PCR purification column, and an aliquot run on the Agilent bioanalyser (DNA 1000 assay) to check the size distribution.     Blunt ending, dA-tailing and adapter ligation of fragmented DNA  Sequencing libraries were prepared using a NEBNext DNA sample preparation master mix 1 kit (New England Biolabs).  Fragmented DNA  was mixed with 10ul Phosphorylation Reaction Buffer (10X), 5ul T4 DNA Polymerase, 4 µl Deoxynucleotide Solution Mix,   5ul T4 Polynucleotide Kinase 1ul DNA Polymerase I, Large (Klenow), and   Sterile H2O to a total volume of 100ul   and incubated in a thermal cycler for 30 minutes at 20°C. End repaired products were purified on a QIAquick column (Qiagen) and eluted in 32 µl of elution buffer. The  dA-Tailing of End Repaired DNA was  carried out by adding 5ul NEBuffer 2 (10X), 10ul Deoxyadenosine 5´-Triphosphate, and 3ul Klenow Fragment (3´? 5´ exo–)  in a  total volume  of  50 µl, followed by  incubation in a thermal cycler for 30 minutes at 37°C. A-tailed DNA was purified down a MinElute® (Qiagen) column and eluted in 10 µl of elution buffer.  Adaptor Ligation of the dA-Tailed DNA was carried out using custom indexed adapters based on Illumina’s Paired- end adapter sequence with the addition of a unique 5- base tag on the 5’ends.  25ul Quick Ligation Reaction Buffer (2X), 1ul custom indexed adaptor (50 µM), and 5ul Quick T4 DNA Ligase was mixed with dA-Tailed DNA in a total volume of 50 µl and incubated in a thermal cycler for 15 minutes at 20°C. Adaptor ligated products were purified on QIAquick columns (Qiagen) and eluted in 32 µl of elution buffer.  Gel purification and size selection of adapter ligated DNA  30ul of purified adapter ligated DNA was run on a 2% agarose gel alongside HyperLadder IV (BIOLINE) at 120V for approximately 2 hrs.  A band equating to approximately 350bp was extracted from the gel and purified using a Qiagen Gel extraction kit following the manufacturers’ instructions, except incubation of the gel piece in QG buffer was performed at room temperature for 5-10 min rather than at 50 °C to help reduce GC-bias in the downstream sequencing data.    Pre-Capture Ligation mediated PCR (LM-PCR)  30ul of adapter ligated DNA library was combined with 50ul Phusion High Fidelity PCR Master Mix (2x), 2ul Pre-Capture PCR Primer PE1 (5'- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGATC* T -3') , 100 µM (final con.:2µM),  2ul Pre-Capture PCR Primer PE2 ( 5'- CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTTCCG ATC* T -3') 100 µM (final con.:2µM), and 16 µl molecular grade water to a final volume of 100µl.  Samples were subjected to 30 seconds at 98°C, followed by 15 cycles of 10 seconds at 98°C, 30 seconds at 65°C, 30 seconds at 72°C, followed by final extension for 5 minutes at 72°C.  Amplified adapter ligated products were purified on QIAquick columns (Qiagen) and eluted in 32 µl of molecular grade water. Samples were quantified by Qubit fluorometer (Invitrogen) using the high sensitivity DNA assay, and an average size distribution of between 300-400bp was verified on the Agilent bioanalyser (DNA 1000 assay).