Oct 05, 2020
Amplicon Library Preparation
- 1Scripps Institution of Oceanography
- A.E. Allen Lab
Protocol Citation: Ariel Rabines, Rob Lampe, Andrew E Allen 2020. Amplicon Library Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bmuck6sw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 29, 2020
Last Modified: October 05, 2020
Protocol Integer ID: 42596
Keywords: 16S, 18S, amplicon, metabarcoding, environmental microbiology,
Abstract
18S and 16S amplicon library preparation protocol.
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.
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 Parada et al. 2015), 18Sv4 (V4F/V4RB primers from Balanzo et al. 2015), and 18Sv9 (1389F/1510R primers from Amaral-Zettler et al. 2009). 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 here.
We also highly recommend including mock communities in every 96-well plate.
Guidelines
Please see the information in the abstract about demultiplexing. You will not be able to properly demultiplex a library using these primers with other software.
Materials
MATERIALS
Agencourt AMPure XPBeckman CoulterCatalog #A63880
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
Quant-iT™ PicoGreen™ dsDNA Assay KitThermo FisherCatalog #P11496
Qubit dsDNA HS Assay KitInvitrogenCatalog #Q32851
TruFi DNA Polymerase KitCatalog #AZ-1710
Setup a 25 µL PCR reaction as follows:
| Reagent | 1x reaction (uL) | |
| 5x buffer | 5.0 | |
| F + R primer combined (10 uM) | 1.0 | |
| DNA polymerase | 0.25 | |
| Template (10 pg - 500 ng) | 1.0 - 2.0 | |
| Molecular grade H2O | to 25 uL |
Run the PCR reaction:
| Step | Temperature (degrees C) | Time | |
| Initial Denaturation | 95 | 1 minute | |
| 30 Cycles | 95 | 15 seconds | |
| 56 | 15 seconds | ||
| 72 | 30 seconds | ||
| Hold | 4 |
Run 2.5 µL of the PCR product on a 2% gel to confirm amplification in all samples except negative controls.
Clean up the PCR products with Agencourt AMPure XP beads with their standard PCR purification protocol and eluting in 35 µL of elution buffer.
Quantify 1 µL of each sample in duplicate with the PicoGreen ensuring that you have a good standard curve and samples have duplicates CV values.
Pool 10 ng purified PCR product. For small libraries, you may want to pool 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.