TITLE: Amplicon Library Preparation
AUTHORS: Ariel Rabines, Rob Lampe, Andrew Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">18S and 16S amplicon library preparation protocol.</div><div class = "text-block">DNA or RNA is usually extracted with our automated protocols from sterivex but other types of samples are occasionally used. If using RNA, first generate cDNA with Invitrogen's SuperScript III First-Strand Synthesis System. Blanks from the nucleic acid extraction should always be used as negative controls. Normally, DNA is diluted 10-fold after extraction and 1 μL of the diluted DNA or the cDNA is used for template in the PCR reaction. If DNA concentrations are very low, 1 μL of the undiluted DNA may need to be used.</div><div class = "text-block"><span>The library is generated with a 1-step PCR, i.e. amplicons are amplified and barcoded simultaneously. Amplicons can be generated for 16S (515F-Y/926R primers from </span><a href="https://doi.org/10.1111/1462-2920.13023" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Parada et al. 2015</span></a><span>), 18Sv4 (V4F/V4RB primers from </span><a href="https://doi.org/10.3354/ame01740" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Balanzo et al. 2015</span></a><span>), and 18Sv9 (1389F/1510R primers from </span><a href="https://doi.org/10.1371/journal.pone.0006372" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Amaral-Zettler et al. 2009</span></a><span>). Barcoded primers for all of these amplicons are attached to this protocol. Typically, the primers are pre-mixed into 96-well plates such that one set of 8 unique F primers (plate rows) and one set of 12 unique R primers (plate columns) are mixed to create 96 unique combinations. Reactions can then be run in a 96 well plate where template plate positions correspond to unique barcode plate positions. The i5 barcode is inline to generate higher sequence quality but also requires a customized demultiplexing workflow. When sequencing, only demultiplex based on the i7 index first. Scripts to then demultiplex based on the i5 index can be found on our Github </span><a href="https://github.com/allenlab/i5_index_demultiplex" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span>. </span></div><div class = "text-block">We also highly recommend including mock communities in every 96-well plate.</div></div>

[STEPS]
?. Setup a  PCR reaction as follows:  AB1Reagent1x reaction (uL)25x buffer5.03F + R primer combined (10 uM)1.04DNA polymerase0.255Template (10 pg - 500 ng)1.0 - 2.06Molecular grade H2Oto 25 uL
25 µl
AB1Reagent1x reaction (uL)25x buffer5.03F + R primer combined (10 uM)1.04DNA polymerase0.255Template (10 pg - 500 ng)1.0 - 2.06Molecular grade H2Oto 25 uL
?. Run the PCR reaction:  ABC1StepTemperature (degrees C)Time2Initial Denaturation951 minute330 Cycles9515 seconds45615 seconds57230 seconds6Hold4
ABC1StepTemperature (degrees C)Time2Initial Denaturation951 minute330 Cycles9515 seconds45615 seconds57230 seconds6Hold4
?. Run  of the PCR product on a 2% gel to confirm amplification in all samples except negative controls.
2.5 µl
?. Clean up the PCR products with Agencourt AMPure XP beads with their standard PCR purification protocol and eluting in  of elution buffer.
35 µl
?. Quantify of each sample in duplicate with the PicoGreen ensuring that you have a good standard curve and samples have duplicates CV values.
1 µl
?. Pool  purified PCR product. For small libraries, you may want to pool  .
10 ng
20 ng
?. Clean and concentrate the final library with XP beads. Elute in  .
45 µl
?. Analyze the final library on the TapeStation and quantify with the Qubit.