DNA seq : 1,4-dioxane anaerobic biodegradation
The V4 hypervariable region of the 16S rRNA gene was amplified using the Illumina compatible, dual indexed primers 515f/806r. These primers and the library construction protocol developed in Patrick Schloss' lab are described in Kozich, JJ et al. (2013)[1]. PCR products were batch normalized using Invitrogen SequalPrep DNA Normalization plates and product recovered from the plate pooled. This pool was cleaned up and concentrated using AmpureXP magnetic beads. The pool was QCd and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Kapa Illumina Library Quantification qPCR assays. The pool was then loaded onto an Illumina MiSeq v2 Standard flow cell and sequencing was performed in a 2x250bp paired end format using a MiSeq v2 500 cycle reagent kit. Custom sequencing and index primers complementary to the 515f/806r oligos were added to appropriate wells of the reagent cartridge as described in ref. [1]. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1.