7weeksBird12
MiSeq sequencing was performed at DNA Services (DNAS) Facility  University of Illinois at Chicago (UIC). Before sequencing, a second PCR amplification was performed in a 10l reaction in 96-well plates. A mastermix for the entire plate was made using the 2X AccuPrime SuperMix II. Each well received a separate primer pair, obtained from the Access Array Barcode Library for Illumina Sequences. The final concentration of each primer was 400nM, and each well received a separate primer set with a unique 10-base barcode (Fluidigm, South San Francisco, CA; Item# 100-4876). Separate reactions with unique barcodes were included for positive control, no-template control (reaction 1), and a second no-template control reaction containing only Access Array Barcode library primers. Cycling conditions were as follows: 95C for 5minutes, followed by 28 cycles at 95C for 30, 60C for 30 and 68C for 30. A final, 7minute elongation step was performed at 68C. PCR yields of positive and negative controls and select samples were validated using Qubit fluorometric quantitation with the Qubit 2.0 fluorometer (Life Technologies) and with size and quantification employing an Agilent TapeStation2200 device with D1000 ScreenTape (Agilent Technologies, Santa Clara, California). After assessing no amplification in the negative controls, samples were pooled in equal volume and purified using solid phase reversible immobilization (SPRI) cleanup, implemented with AMPure XP beads at a ratio of 0.6X (v:v) SPRI solution to sample. This ratio removes DNA fragments shorter than 300bp from the pooled libraries. The final quality control was performed using TapeStation2200 and Qubit analysis, prior to dilution to 6pM for emulsion PCR. Pooled, diluted libraries were sequenced on an Illumina MiSeq instrument, using a MiSeq 600-cycle sequencing kit version 3, and analyzed with Casava1.8 (pipeline 1.8). Reads were 200 nucleotides in length (paired end, 2200). PhiX DNA was used as a spike-in control. Barcode sequences from Fluidigm were provided to the MiSeq server, and sequences were automatically binned according to 10-base multiplex identifier (MID) sequences. Raw reads were recovered as FASTQ files.