Chinese spring SOLiD 4 sequencing
Triticum aestivum cv. Chinese spring SOLiD 4 sequencing
Library construction followed methods in the Applied Biosystems SOLiD 5500 Series Library Preparation Guide, as this improved on the SOLiD 4 protocol. For Emulsion PCR, templated beads were prepared from the mate-paired library using the ePCR kit v3 and the Bead Enrichment Kit from Applied Biosystems for SOLiD 5500 and the SOLiD™ EZ Bead™ System. A Workflow Analysis was carried out after the first round of templated bead preparation using the Workflow Analysis kit from Applied Biosystems to check library quality and the amount of templated beads generated per ePCR. Templated beads were deposited on slides using the Bead Deposition kit from Applied Biosystems and sequencing was done according to the Applied Biosystems SOLiD 4 System Instrument Operation Guide using the reagents from Applied Biosystems
Triticum aestivum
A 2x50bp Mate-paired library was constructed following the Applied Biosystems 5500 Series SOLiD™ System Library Preparation Guide. 5μg of genomic DNA were fragmented by HydroShear (Digilab Genomic Solutions Inc) to 2-3.5 kb. Fragmented DNA was run on a 1% Agarose gel and DNA fragments between 2-3 kb were selected, and subsequently end-repaired. MP adaptors were ligated to the sheared end-repaired DNA. Fragments were circularized by intra-molecular hybridization where the mate paired adaptors contain a blocking oligonucleotide to protect the 3’ overhangs of the MP Adaptors from self-annealing. Plasmid-Safe DNase was used to eliminate the un-circularized DNA and 1.3 μg of circularized DNA was recovered after purification using Agencourt Ampure XP beads. Nick translation using E. coli DNA polymerase was performed for 10 minutes at 5°C to produce mate-paired tags of 100bp. The nick translated DNA was digested with T7 exonuclease and S1 nuclease to cleave the mate-paired tags from the circularized template. P1-Tand P2-T adaptors were ligated to the fragments to generate the 250-350 bp library. This was then amplified using Library Primers 1 and 2 with Platinum Amplification Mix and 9 cycles of amplification.
UOL_CGR
Chinese spring SOLiD 4 sequencing