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posted on 30.11.2018, 03:47 by Kendra Abts, J. Andrew DeWoody, Jamie Ivy
Genomic DNA was extracted from whole blood or tissue samples of 52 koalas. DNA concentrations were quantified using a Nanodrop spectrophotometer and stock solutions were standardized at 5 ng/uL, with the exception of individual (ZIMS# 501277), which had a concentration of 2.5 ng/uL. We amplified primer sets previously characterized from the expressed MHC transcripts in three SDZ koalas [Abts et al. 2015], and 4 additional primer sets from Lau et al. 2013. We began with eleven primer sets: DAA, DAB, DBA, DBB, DCB, DMA, DMB, UAB1, UB1, UE, and UK, named according to the guidelines set forth in Klein [1990]. Each set of PCR primers was optimized and used to amplify the target regions and each individual was amplified twice at each primer set in a modified Lenz-Becker approach to minimize artifact alleles. Barcoded TruSeq front-end adaptors then were added to the amplicons to uniquely identify each replicate. The products were then quantified using a Qubit fluorometer and pooled in equal concentrations of approximately 10 nM. Then the pool was sequenced using 2x300bp chemistry and an Illumina MiSeq sequencer. The reads from each replicate were separated using the barcoded front-end adaptors. These reads are arranged by individual-replicate (indicated by the three digits - one digit after the initial six digit unique identifier). R1 or R2 represents the forward and reverse reads, respectively.









Phascolarctos cinereus