TITLE: Library Preparation of Bee 18S and 28S rRNA amplicons for High-Throughput Illumina Sequencing
AUTHORS: Brian Darby, Russ Bryant, Abby Keller, Madison Jochim, Josephine Moe, Zoe Schreiner, Carrie Pratt, Ned H. Euliss Jr., Mia Park, Rebecca Simmons, Clint Otto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The objective of this protocol is to prepare amplicon sequencing libraries for high-throughput sequencing (via Illumina MiSeq) of the 18S SSU rRNA and 28 LSU rRNA loci for molecular identification of bee communities. </div></div>

[STEPS]
?. [Sample preparation and gDNA extration]
Place one mesothoracic leg from each specimen (of a sample) into a 2.0 ml microcentrifuge tube, and repeat this with a new microcentrifuge tube for all samples that are to be separately barcoded.
?. [First Round PCR Amplifcation]
Prepare Master Mix 1 for each locus separately (18S and 28S) for first-round PCR amplification:  ABC1Volume (per reaction)Working stock concentrationReagent210 ul2XDreamTaq MasterMix30.4 ul10 uM18S FOR primer BD0150*40.4 ul10 uM18S REV primer BD0151*58.2 ulMolecular grade water619 ul Total MasterMix1
ABC1Volume (per reaction)Working stock concentrationReagent210 ul2XDreamTaq MasterMix30.4 ul10 uM18S FOR primer BD0150*40.4 ul10 uM18S REV primer BD0151*58.2 ulMolecular grade water619 ul Total MasterMix1
?. [Sample preparation and gDNA extration]
Add five ceramic beads and 500 ul genomic lysis buffer from the desired DNA extraction kit (e.g. Qiagen 96-well Tissue and Cells) to each tube. Optional: and two mealworm beetle (Tenebrio molitar) legs as inter-sample controls.
?. [Sample preparation and gDNA extration]
Lyse tissue at room temperature overnight, then pulverize for 10 min in a TissueLyser prior to proceeding with the desired genomic DNA extraction kit instructions (e.g. Qiagen 96-well Tissue and Cells kit). Elute DNA into 60 ul elution buffer, pH 8.0.
?. [First Round PCR Amplifcation]
For each locus separately, add 19 ul of Master Mix 1 and 1 ul of gDNA (10 ng/ul) to a suitable PCR reaction tube (or well in a 96-well plate).
?. [First Round PCR Amplifcation]
Amplify first round PCR reactions in a thermocycler with 30 cycles of 95 °C for 0:30, 55 °C for 0:30, and 72 °C for 0:30, followed by a final extension at 72 °C for 5 minutes.
?. [First Round PCR Amplifcation]
Keeping each sample separate, pool 10 ul of PCR product from each locus (e.g. 18S and 28S), and purify with a PCR clean-up kit (e.g. Zymo 96-well PCR CLeanup kit).
?. [Second Round PCR Amplification]
For each sample, add reagents to an appropriate PCR reaction tube (or well of a 96-well plate):  ABC1Volume (per reaction)Working stock concentrationReagent210 ul2XDreamTaq MasterMix32 ul2 uMFOR Nextera barcoded primer*42 ul2 uMREV Nextera barcoded primer*55 ulMolecular grad water62 ulpurified 1st-round PCR product720 ul Total reaction volume
ABC1Volume (per reaction)Working stock concentrationReagent210 ul2XDreamTaq MasterMix32 ul2 uMFOR Nextera barcoded primer*42 ul2 uMREV Nextera barcoded primer*55 ulMolecular grad water62 ulpurified 1st-round PCR product720 ul Total reaction volume
?. [Second Round PCR Amplification]
Amplify second round PCR reactions in a thermocycler with 8 cycles of 95 °C for 0:30, 55 °C for 0:30, and 72 °C for 0:30, followed by a final extension at 72 °C for 5 minutes.
?. [Second Round PCR Amplification]
Pool 5 ul of second-round PCR product from each sample and clean half of this using a PCR cleanup kit (e.g. Zymo PCR Cleanup kit). Elute in 30 ul elution buffer (EB), pH 8.0, and this eluted product is ready to submit for cluster formation on the Illumina MiSeq (using 2x300 bp paired end reads).
?. [Second Round PCR Amplification]
For best results, gel purify a large portion of the cleaned second-round PCR products to target the >500 bp bands and minimize