title: S9-Shotgun-G12-calc
design: "Library construction for S9 extract of G12. General information: Illumina libraries were built for nine ancient DNA extracts prepared from 50 mg dental calculus samples from individuals B61 and G12. Target DNA quantity for each library preparation was 500-700ng. For those extracts with low DNA concentration, all of the available extract was used. A negative extraction control was processed alongside the experimental extracts and monitored for contamination. No contamination was observed. In an ancient DNA clean room, DNA extracts were repaired using a NEBNext End Repair and dA-Tailing kit following manufacturer?s instructions. Short Illumina Indexed PE Adaptors were added to the extracts as follows: 1ml of 15mM adaptor for libraries S2, S5, S6, S7, and S8 (high concentration extracts), and 1ml of 5mM adaptor for libraries S1, S3, and S9 (low concentration extracts). A unique, 6-nucleotide index was used for each library, and each index was distinguished by at least two differences from other indexes. Quick T4 Ligase (1ml) was added to each library and incubated at 25?C for 10 min. After adaptor ligation, the libraries were purified on a MinElute spin column (Qiagen) and eluted into 20ml EB buffer. The libraries were then PCR-amplified in two rounds to increase concentration and complete adapter assembly. At the end of library construction, the total length of the indexed adaptors is 122bp. PCR inhibition was observed during the first amplification step, and the following PCR reactions were optimized by sample to yield the best amplification. The first PCR reaction was set up as follows: 25ml Phusion HF Master Mix (Finnzymes), 2ml 10mM forward PE primer, 2ml 10mM reverse indexed PE primer, 13ml H2O. For libraries S1, S3 and the negative extraction control, 8ml library was added to the reaction. For libraries S2, S4 and S9, 1ml library and 7ml H2O were added. Library amplification for S2 and S4 was repeated with 1ml library, 1ml BSA (2.5mg/ml) and 6ml H2O. For libraries S5-S8, 1ml of diluted 1:9 libraries and 7ml H2O were added. PCR tubes were then sealed and transferred to a PCR facility. Cycling conditions were: 98?C for 60s, 12 cycles of (98?C for 20s, 60?C for 30s, 72?C for 30s), and a final extension of 72?C for 5 min. PCR products (4ml) were visualized on a 2% agarose gel to confirm amplification and purified on a MinElute spin column (Qiagen) and eluted into 20ml EB buffer. Amplification products were observed for all nine archaeological calculus samples, but no amplification was observed in the negative extraction control. A second PCR reaction was set up as follows: 12ml Phusion HF Master Mix (Finnzymes), 1ml 10mM forward PE primer, 1ml 10mM reverse indexed PE primer, 1ml H2O, and 8ml library. Cycling conditions were: 98?C for 120s, 8 cycles of (98?C for 20s, 60?C for 35s, 72?C for 30s), and a final extension of 72?C for 5 min. PCR products (4ml) were visualized on a 2% agarose gel to confirm amplification and purified on a MinElute spin column (Qiagen) and eluted into 20ml EB buffer. Library DNA concentration was measured with a Qubit DNA High-Sensitivity Assay Kit (Invitrogen) and the duplicate S2 and S4 libraries were pooled. Concentration and size distribution of the libraries was determined on a Bioanalyzer 2100 DNA 1000 chip (Agilent Technologies). Mean library length ranged from 193 bp (S9) to 325 bp (S8), which, after subtracting the adaptor length of 122bp, indicates a mean extract insert length of 71-203bp. This length is consistent with ancient DNA, which is known to be highly fragmented. DNA concentration was further characterized using a qPCR SYBRGreen Assay, and the nine libraries were pooled at equimolar concentrations. Following cluster generation, libraries were sequenced on one lane of an Illumina HiSeq 2000 in a 100 bp, single-end run, followed by base-calling using standard Illumina software (RTA version 1.12.4.2). Correctly indexed reads (100% index identity) were then filtered and binned using Illumina?s CASAVA software (version 1.8.0). Reads containing Ns were trimmed, as were bases with Phred quality scores <35. Adapter sequences were removed using the AdapterRemoval tool, and reads <25bp were discarded. The remaining reads were verified using fastqc analysis."