Multiplex Barcode Sequencing

by Rasmus Bojsen and David Gresham

Overview

The protocol generates a single library for sequencing on an Illumina HiSeq using a  50bp single end run.  The protocol has been designed to multiplex 120 separate samples.  The sequencing library is generated in three steps:

  1. In the first step, UPTAGS are amplified from a pool of mutants using PCR1, which introduces a unique 5 base pair sample index (e.g. Sample1=>Index1=>ATACC). DNTAGS from the same sample are amplified using DNTAG specific primers that introduce the same sample index (e.g. for DNTAGs Sample1=> Index1=>ATACC).
  2. In the second step all UPTAG libraries from up to 120 different samples, each with a unique 5bp index, are pooled and the complete Illumina adaptor is generated using PCR.  In a separate PCR, all DNTAG libraries from up to 120 different samples, each with a unique 5bp index, are pooled and the complete Illumina adaptor is generated using PCR.
  3. The multiplexed UPTAG libraries and multiplexed DNTAG libraries are mixed together in equal amounts and sequenced on an Illumina flow cell using the standard Illumina sequencing primer and 50bp single end read.

The final PCR products that are sequenced on the Illumina HiSeq are a 1:1 mixture of UPTAG and DNTAG molecules as follows:

UPTAGsUPTAG_Forward_Read

DNTAGsDNTAG_Forward_Read

 

PCR 1: Index incorporation

The aim of the first PCR is to amplify the molecular barcodes from genomic DNA and incorporate a unique 5bp index (see file IndexTagSpecs.xls). This PCR incorporates a 15 base sequence at the 5’ end of the molecule for subsequent addition of the Illumina adaptor and complete sequencing primer sequence and incorporates the full length of the Illumina adaptor at the 3’ end of the molecule.

Each barcode has a unique UPTAG and DNTAG. For each sample these are independently amplified using primers that share a common sample index, which denotes the sample ID. For each sample, the UPTAGS and DNTAGS have a unique PCR priming site, which is sequenced immediately after the index, allowing identification of the read as either an UPTAG (5’-GTCCACGAGGTCTCT-’3) or a DNTAG (5’-GTGTCGGTCTCGTAG-3’) for each sample (e.g. sample1=> index1=>ATACC).

UPTAG PCR (UPTAG Primer sites in green, 5bp sample index underlined)

Primers:
Illumina UPTAG Index 1: 5’-ACGCTCTTCCGATCTATACCGTCCACGAGGTCTCT-3’
Illumina UPkanMX: 5’-CAAGCAGAAGACGGCATACGAGATGTCGACCTGCAGCGTACG-3’

 

Template: Genomic DNA from barcoded mutants
Generates the following molecule:
UPTAG_PCR

 

DNTAG PCR (DNTAG Primer sites in purple, 5bp index is underlined)

Primers:
Illumina DNTAG Index 1: 5’-ACGCTCTTCCGATCTATACCGTGTCGGTCTCGTAG-3’
Illumina DNkanMX: 5’-CAAGCAGAAGACGGCATACGAGATACGAGCTCGAATTCATCG-3’

 

Template: Genomic DNA from barcoded mutants
Generates the following molecule:
DNTAG_PCR
PCR mix

Component 1x Master Mix (x MM)
5x buffer 5 µl 5 µl x MM
dNTP (2.5 µM) 2.5 µl 2.5 µl x MM
Taq (Prime star) 0.2 µl 0.2 µl x MM
Illumina UP/DN (10 µM) 2 µl 2 µl x MM
Illumina UPTAG Index1 2 µl
dH2O 10.3 µl 10.3 µl x MM
Template 2 µl
Total 24 µl

 

When performing multiple PCRs:

  • Use an Eppendorff repeater electronic pipette to dispense 20 µl PCR mix to PCR tubes
  • Use multichannel pipette to distribute 2 µl Index primer (10 µM) from microtiter plate stock to PCR tubes
  • Add 2 µl DNA (50 µg/ml) sample to PCR tubes
  • Final PCR volume is 24 µl

PCR program (BARSEQ_STEP1) (App. time: 45 min)

  1. 2 min at 98°C
  2. 10 sec at 98°C
  3. 10 sec at 50°C
  4. 10 sec at 72°C
  5. Go to step 2. Repeat 20 times
  6. 2 min at 72°C
  7. Infinite at 4°C

Add loading dye to clean PCR tubes. Use the multichannel pipette to transfer 1µl loading dye to parafilm. Mix loading dye with 5µl PCR product using the multichannel pipette and load into a 26 well 2 % agarose gel. Use 3 µl of 100 bp ladder. Run for 45 min at 50V (see file Index_primer_overview.pdf).

In order to estimate the DNA concentration of each PCR product measure gel band intensity and estimate the DNA concentration using the equation:
DNA_Conc_Fit

PCR2: Illumina P5 incorporation

The aim of the second PCR is to incorporate the Illumina P5 sequence. All UPTAG and DNTAG PCR1 reactions can be pooled into a single UPTAG and a single DNTAG pool and used as the two templates for PCR2. The same reverse primer is used in PCR2 as was used in PCR1. 

UPTAGs

Primers:
P5: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
IlluminaUPkanMX: 5’-CAAGCAGAAGACGGCATACGAGTCGACCTGCAGCGTACG-3’

Template: Pool of normalized PCR1 UPTAG products

PCR reaction:
UPTAG_Adaptor

 

DNTAGs

Template: Pool of normalized PCR1 DNTAG productsPCR reaction:
DNTAG_AdaptorProcedure:

  • Make a mixture of equimolar amounts of PCR1 UPTAGS and a mixture of equimolar amounts of PCR1 DNTAGS (i.e. by combining 60 ng of each library)
  • Perform a column-based PCR clean up

Incorporate the Illumina P5 sequence to the pooled DNTAGS and UPTAGS:

  • 5 µl 5x buffer
  • 5 µl dNTP (2.5 mM)
  • 2 µl Taq (Prime star)
  • 2 µl Illumina UP/DN (10 µM)
  • 2 µl Illumina P5 (10 µM)
  • 2 µl DNA template (UP/DN pool)
  • 3 µl dH2O

PCR program (BARSEQ_STEP1) (App. time: 45 min)

  • 2 min at 98°C
  • 10 sec at 98°C
  • 10 sec at 50°C
  • 10 sec at 72°C
  • Go to step 2. Repeat 20 times
  • 2 min at 72°C
  • Infinite at 4°C

Run PCR product on a 2% agarose gel for 45 min at 50V. Include PCR product from PCR1 as control (see file Index_primer_overview.pdf).Library qPCR quantification and submissionPool the P5 incorporated UPTAG and DNTAG pools in equimolar amount.Dilute to 5 ng/µlLibrary qPCR quantificationDilute to 2 nM in 25 µl volumeSubmit samples for 50bp SE sequencingimage1