TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Illumina® short-read DNA sequencing has become an integral tool in biology for genome-wide studies. Offering accurate base-pair resolution at the most competitive price, the technology has become widespread. However, the generation of multiplexed DNA libraries remains costly and cumbersome. Here, we present a streamlined cost-conscious protocol for generating multiplexed short read DNA libraries using a transposase from Illumina®. By implementing small volumes that use 1/25th the amount of transposase compared to Illumina® NexteraTM protocols, the cost of library preparation can be significantly reduced, by 1/10th or more. Furthermore, we optimised the protocol to minimise carboxylate bead-based cleanups between steps, further reducing cost, time and DNA input. By developing our own indicies to multiplex nine 96-well plates, up to 864 samples can be placed on a single flow cell. This enables efficient usage of monolithic sequencing platforms that can offer over three terabases of sequencing per flow cell.

[BEFORE_START]
In this protocol, the transposase being used will be diluted 1/25, therefore up to 25 times more reaction buffer is necessary. Prepare the custom buffer 2x TMgCl2: 20 mM Tris-HCl (pH 8.4-9), 10 mM MgCl2. Recommended setup:

   Component 
   Stock 
   Quantity (1 mL) 
     20 mM Tris-HCl (pH8.4-9) 
   500 mM 
   40 µL 
     10 mM MgCl2 
   250 mM 
   40 µL 
     Nuclease-free water 
   N/A 
   920 µL 
  
 

   Materials
 
 96-well half skirt PCR microplate (Axygen Scientific® PCR-96M2-HS-C).
 96-well PCR microplate white, Roche 480 light cycler (Axygen Scientific® PCR-96-LC480-W-NF) (or an alternative microplate for fluorescent readings).
 384-well plate (4titude FrameStar 4ti-0384/B).
 PCR-grade adhesive film for microplates (e.g. Eppendorf storage film 0030127870).
 2 mL SC micro tube PCR-PT (Starstedt 72694.406).
 1.5 mL Eppendorf tube. 
 10 or 15 mL Falcon tube.
 MinElute® PCR purification kit (Qiagen 28004).
 Agencourt AMPure XP (Beckman Coulter A63881).
 Quant-iTTM dsDNA assay kit, high sensitivity (Molecular ProbesTM Q33120).
 NexteraTM tagment DNA enzyme 1 (TDE1) (Illumina® catalogue # 15027865, also available in Illumina® kits FC-121-1030* or FC-121-1031*).
 Q5® Hot Start High-Fidelity DNA Polymerase (New England BioLabs® M0493).
 10 mM dNTPs (New England BioLabs® N0447).
 Custom barcode oligos (see supplemental), or Illumina® NexteraTM index kit.
 70% ethanol (analytical grade or higher).
 Nuclease-free water.
 
*Illumina® has issued a product obsolescence notification for these kits. However, TDE1 can ordered as a standalone component. This protocol has not tested the bead-bound transposase in the new Illumina® NexteraTM DNA flex kits. However, it is likely minimal to no optimisation would be required. For instance, magnetizing an aliquot of the bead-bound transposase and resuspending in 2x TMgCl2.
  
  
 Equipment
 
 JANUS automated workstation, such as NGS express or G3 (PerkinElmer®) (recommended but not essential).
 JANUS automated workstation accessories including; reagent reservoirs, plate adaptor support tiles, 25 and 175 µL filtered tips (PerkinElmer®).
 Infinite® M1000 PRO microplate reader (Tecan).
 PCR machine for 96-well plate.
 PCR machine for 384-well plate or a microplate heat block.
 Magnet rack for PCR tubes or 1.5 mL Eppendorf tubes. 
 Qubit Fluorometer (ThermoFisher Scientific). 
 High sensitive electrophoresis system; e.g. GXII Labchip (Perkin Elmer®) or Bioanalyzer (Agilent).
 (Optional) Automated agarose gel electrophoresis system, e.g. Pippin Prep (Sage Science).


[GUIDELINES]
This protocol is designed for Illumina® short-read sequencing and requires previously purified DNA. The DNA will be diluted and later amplified, thus only a small input is needed, as little as 2.24 ng. To aid conducting this protocol, program files for JANUS automated workstations (PerkinElmer®) have been provided in the supplemental with descriptions and a list of compatible hardware. Helpful excel sheets to create standard curves for DNA quantification and dilution strategies are also provided. Lastly, custom indicies are provided to multiplex nine unique 96-well plates, enabling multiplexing up to 864 samples on a single sequencing run.

 

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SECTION: DNA quantification
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SECTION: DNA dilution
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SECTION: Tagmentation
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SECTION: PCR for enrichment and barcoding
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SECTION: PCR for enrichment and barcoding
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SECTION: Pool libraries
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SECTION: Pool libraries
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Initial clean: MinElute® columns
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SECTION: Optional size selection: Pippin Prep
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SECTION: Optional size selection: Pippin Prep
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SECTION: Optional size selection: Pippin Prep
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Final clean: AMPure XP beads
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SECTION: Preparation for sequencing
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SECTION: Preparation for sequencing
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