title: SR-GS of MDS patients
design: The samples are sequenced on NovaSeq platform, libraries of 450-600 bp insert size will be prepared using KAPA Hyper Prep Kit. DNA (750 ng) is sheared into fragments of approximately 450-600 bp in a Covaris E220 system (Covaris, Inc. Woburn, MA) in batches of 96 samples at a time, followed by purification of the fragmented DNA using the AMPure XP beads, double SPRI bead clean up with different ratios of AMPure XP beads (0.57X/ 0.75X), to select a narrow band of sheared DNA for library preparation. Using KAPA Hyper kit (KK8505), DNA end-repair and 3'-adenylation are performed in the same reaction followed by ligation of the barcoded adaptors to create PCR-Free libraries. A set of 96 8-bp barcoded adapters (Illumina TruSeq UD Indexes v2, # 20040870) are currently in use for sample barcoding. Post ligation products are cleaned twice with 0.8X AMPure XP beads to get rid of leftover adapters/adapter dimers that may otherwise interfere with sequencing. The final library size estimation and quantification are done using the Fragment Analyzer (Agilent, Inc) electrophoresis system and Quant Studio™ 6 Flex Real-Time PCR System (Applied Biosystems™) respectively to achieve an average final library size of ~530bp and must be greater than 470 bp with a minimum concentration >1.8nM. The NovaSeq instrument is employed for sequencing to generate 150 bp, dual indexed and paired-end sequence reads in a format of multiplexed pools to generate ~110 GB per sample to achieve 30X.