The best option for sequencing larger numbers of amplicons that exceed the MiSeq read length limitations (e.g. amplicons longer than 600 nt) is Pacbio sequencing.
Amplicons up to 12 kb length can generate highest quality sequence data for individual molecules employing circular-consensus-sequencing (CCS) analysis. Amplicons longer than 3.5 kb should be run with 20 hour movies (sequencing run times). Long Amplicon Analysis (LAA) can generate highest quality consensus data for amplicons of up to 10 kb length from reads of multiple library molecules. Please see this poster for the principles (although the read metrics are very outdated).
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The most efficient way to mulitplex amplicon samples for Pacbio sequencing employs a two-step PCR:
- The first PCR is carried out with sequence-specific oligos that are tagged on the 5′-ends with universal tags. The sequences are provided below.
Please note that these first-round primers are amino-modified at the 5′-end to prevent the conversion of unbarcoded amplicons into library molecules. Ordering desalted oligos should be sufficient, as long as the sequence-specific part of the oligo is short (e.g. 20 bp). - The second PCR then uses the universal tags to add sample-specific 16 bp barcode indices to both ends of each amplicon.
It is highly recommended that you carry out these PCRs in your lab, as well as the equimolar pooling of the amplicons. We will then carry out a single Pacbio library prep and could sequence the pool on a single Pacbio SMRT-cell.
We can provide a set of barcoded PCR indices with the universal tags described below. We have two sets of 12×12 and 24×24 (forward and reverse) indices available, allowing for the pooling of up to 576 samples. The sequences of our PacBio barcode set are available here: PacBio-PCR-Barcodes-24×24-Set-2018
Please contact our PacBio specialist Oanh Nguyen at ohnguyen@ucdavis.edu .
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The suggested protocol is described in detail for full length 16S sequencing here: Full-Length-16S-Pacbio-Protocol
Please modify this protocol by substituting your sequence-specific sequences for the 16S-specific sequences (the sequences in upper case on page 1). We suggest to check the first round PCR oligo sequences on the IDT oligoanalyzer (https://www.idtdna.com/calc/analyzer) for secondary structures. It is best to avoid any sequences that generate a Delta G smaller than -9 for any of the structures. Please optimize the conditions of the first PCR to avoid primer-dimer generation. Upon verifying that the PCR products for all samples are clean and of expected size (via agarose gel electrophoresis), the samples should be pooled equimolarly. We suggest to quantify the samples via fluorometry (Qubit, Promega Quantus, or plate reader) for accurate pooling.
Please also see pages 1 to 6 of this information for additional sample preparation advice: Pacbio-Amplicon-Template-Information
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The sequences of the Universal Tags for the adapters are given below and can also be found on this page: https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Barcoding
FORWARD Tag (U1) 5’-/5AmMC6/GCAGTCGAACATGTAGCTGACTCAGGTCAC-3’
REVERSE Tag (U2) 5’-/5AmMC6/TGGATCACTTGTGCAAGCATCACATCGTAG-3’
REVERSE Tag (U2) 5’-/5AmMC6/TGGATCACTTGTGCAAGCATCACATCGTAG-3’
The suggested 16 nt barcode sequences themselves are uploaded here: PacBio_384_Barcode_Sequences.
(originally from here: https://github.com/PacificBiosciences/Bioinformatics-Training/blob/master/barcoding/pacbio_384_barcodes.fasta ).
PacBio uses the reverse complements of the listed barcodes for addition to the REVERSE tags. This barcode set was designed to be used symmetrically (same barcode on either end of insert) or asymmetrically (different barcodes on either end).
For amplicons of up to 5kb length, please submit 1 ug or more of your pooled amplicon sample (in EB buffer) to us for the PacBio library preparation.
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