high GC library
We ordered four ultramers from Integrated DNA Technologies: BC1-BC1, BC2-BC2, BC3-BC3 and the adapter (Table \ref{tableoligos}). BC1-BC1, BC2-BC2 and BC3-BC3 contain the Illumina P5-SBS3T sequence followed by a 20nt barcode, the Lambda phage Attachment site L (AttL) sequence and another 20nt barcode. Whereas the barcodes of BC1-BC1 and BC2-BC2 have a balanced base composition, BC3-BC3 has GC rich barcodes with an expected GC content of $80\%$. The adapter oligonucleotide is $5^\prime$ phosphorylated and contains a 15nt barcode followed by the reverse complement of the Illumina P7-SBS8 sequence. It's $3^\prime$ end is phosphorylated to avoid circularization. We pooled BC1-BC1 and BC2-BC2 in equal proportions and ligated them to the $3^\prime$ adapter using CircLigase ssDNA ligase (epicentre; previously sold as Thermophage ssDNA ligase) as previously described \cite{listructure-independent2006}. For the high GC dataset ZL053 we additionally included BC3-BC3 in the ligation reaction at a fivefold reduced concentration relative to BC1-BC1 and BC2-BC2. The ligation reaction was cleaned up with Agencourt RNAClean XP beads (Beckman Coulter) according to the manufacturers instructions. The ligated products were subjected to 25 cycles of PCR using $47\mu l$ Accuprime Pfx SuperMix (Invitrogen), $1\mu l$ of $10\mu M$ forward and reverse primers each (Table \ref{tableoligos}) and $1\mu l$ input. Cycling was performed in a BioRad MyCycler Thermal Cycler using standard Accuprime protocol with $58^\circ C$ annealing temperature and 30 seconds extension time. The PCR product was gel extracted and sequenced on a single lane of a HiSeq 2000 machine at PE101 per dataset.