TITLE: Workflow for SNP genotyping using the Hi-Plex method
AUTHORS: Anne-Laure Besnard, Daniel J. Park, Bernard J. Pope, Fleur Hammet, Sophie Michon-Coudouel, Marine Biget, Stacy A. Krueger-Hadfield, Stéphane Mauger, Eric J. Petit

[DESCRIPTION]
Many research questions in ecology and evolution require balancing sampling strategies between their spatial (how many populations? on which geographical, environmental gradients?), temporal (diachronic approaches), and genomic (how many and which loci?) dimensions. High-throughput molecular biology protocols often offer very good genomic coverage, but this is often only achievable at the expense of other sampling dimensions. This has led to the development of targeted genotyping strategies for SNP locus sets, in addition to whole or reduced genome sequencing strategies. We here present an adaptation of a protocol developed by the University of Melbourne for genotyping rare variants in human oncology to non model species for use in ecology and evolution. Hi-Plex is an amplicon sequencing technique (sensu Meek & Larson 2019) in which all loci are co-amplified in a multiplex reaction before Illumina or Ion Torrent sequencing (we used Illumina). Intermediate steps include dual indexing of individual samples used for demultiplexing.

[BEFORE_START]
Select a set of SNP loci to include in your SNP panel. Prepare a fasta file with one 145bp sequence per locus. SNP loci can be selected from any genomic resource with information on polymorphism. If such resources do not exist, they can be generated from various protocols (see for instance Delord et al. 2018).
The number of samples to be genotyped should ideally be a multiple of 96
Choose a sequencing technology that allows reaching 100x read depth per locus per sample

[GUIDELINES]
The Hi-Plex protocol is adapted for population genetic studies using SNPs or microhaplotypes. It is an amplicon sequencing technique (sensu Meek & Larson 2019) in which all loci are co-amplified in a multiplex reaction before Illumina or Ion Torrent sequencing (we used Illumina). Depending on the number of loci and samples, and given actual reagent and sequencing costs, multilocus genotypes at up to 400 loci can be obtained for about 10 to 15€ per sample.


[STEPS]
SECTION: Gene Specific Primers (GSPs) and adaptator (TSITs) preparation
2. Gene Specific Primers (GSP) preparation

GSP synthesis

Prepare a fasta file with 145bp sequences that will be used to design GSPs. Sequence names should contain the position and polymorphism of the targeted SNP.
An example of one such sequence is:
>149126:289:-|73|[A/G]|LP_LF_hyb
CGGGCCGGCCCGTCCAAAAATAGCAAGCAGAGTGAAATCCTCCACATTCTAAATGGCCACATGCACAAACAAGCAGTGTTTTCTTAAAGAGACCACTGCTCCCAGCTCACCCAGCAGCCTCTCACAGACAGTGGTGGGGGGGGGG

Send the file to Daniel Park (djp@unimelb.edu.au) and Bernie Pope (bjpope@unimelb.edu.au) for primer design.

Add the following common heels to forward and reverse specific primers:
Forward heel in 5'-3' : CTCTCTATGGGCAGTCGGTGATT
Reverse heel in 5'-3' : CTGCGTGTCTCCGACTCAG

Then, order GSPs in 96-well plates with a minimum yield of 10 nmol, purified using standard desalination with a purity of up to 70% to obtain 100 µL of 100-200 µM concentration in TE.

GSP pool preparation

Prepare 40 µM plates from each 100 or 200 µM GSP plate using 1X low TE (10 mM Tris pH8.0/0.1mM EDTA):
 

Pool all forward and reverse GSPs.
First take 5 µL of each GSP-F and GSP-R at 40 µM and pool them by row in a 8-tube strip.
Then, pool the content of the strip into a 2 mL microcentrifuge tube. See diagram and table below.
Prepare as many aliquots as possible to avoid multiple freeze-thaw cycles.

  
                 Ci GSP (µM)                 Cf GSP (µM)       Ci * Vi/ Vf pool         Vi GSP (µL)                 Number of GSPs       Nb SNP F + Nb SNP R         Vf pool (µL)       Vi GSP * Nb GSP         Cf pool (µM)       Cf GSP * Vf pool         Nb aliquot 100 µL       Vf pool/100 µL            50 SNP - GSP pool         40         0.400         5         100         500         200         5            96 SNP - GSP pool         40         0.208         5         192         960         200         9.6            192 SNP - GSP         40         0.104         5         384         1920         200         19.2            288 SNP - GSP pool         40         0.069         5         576         2880         200         28.8            384 SNP - GSP pool         40         0.052         5         768         3840         200         38.4      
 
Prepare one tube of GSP-pool at 10 µM for PCR 1 in the Hi-Plex protocol.
       Nb of 96-well PCR plates         C pool undiluted    (µM)         C pool diluted    (µM)         Vi    (µL)         Vf    (µL)         V TE    (µL)            1         200         50         12.5         50         37.5            2         200         50         25         100         75            3         200         50         37.5         150         112.5            4         200         50         50         200         150      
The tubes and plates should be stored at -20 ˚C.
SECTION: Gene Specific Primers (GSPs) and adaptator (TSITs) preparation
3. Adaptator (TSITs) preparation

TSITs synthesis 

Order TSIT adaptors in two 96-well plates, one named "TSIT A" (these include the P5 sequence),  and the other named "TSIT P" (these include the P7 sequence), with a purity of at least 80% in 100 µL at 200 µM concentration in TE. A minimum yield of 40 nmol is required. The list of TSITs and the plate maps are given below.

 
            Plate   well   Name   Sequence      A1   N701_TSIT_P   CAAGCAGAAGACGGCATACGAGATTCGCCTTActccgctttcctctctatgggcagtcggtgat      A2   N702_TSIT_P   CAAGCAGAAGACGGCATACGAGATCTAGTACGctccgctttcctctctatgggcagtcggtgat      A3   N703_TSIT_P   CAAGCAGAAGACGGCATACGAGATTTCTGCCTctccgctttcctctctatgggcagtcggtgat      A4   N704_TSIT_P   CAAGCAGAAGACGGCATACGAGATGCTCAGGActccgctttcctctctatgggcagtcggtgat      A5   N705_TSIT_P   CAAGCAGAAGACGGCATACGAGATAGGAGTCCctccgctttcctctctatgggcagtcggtgat      A6   N706_TSIT_P   CAAGCAGAAGACGGCATACGAGATCATGCCTActccgctttcctctctatgggcagtcggtgat      A7   N707_TSIT_P   CAAGCAGAAGACGGCATACGAGATGTAGAGAGctccgctttcctctctatgggcagtcggtgat      A8   N708_TSIT_P   CAAGCAGAAGACGGCATACGAGATCCTCTCTGctccgctttcctctctatgggcagtcggtgat      A9   N709_TSIT_P   CAAGCAGAAGACGGCATACGAGATAGCGTAGCctccgctttcctctctatgggcagtcggtgat      A10   N710_TSIT_P   CAAGCAGAAGACGGCATACGAGATCAGCCTCGctccgctttcctctctatgggcagtcggtgat      A11   N711_TSIT_P   CAAGCAGAAGACGGCATACGAGATTGCCTCTTctccgctttcctctctatgggcagtcggtgat      A12   N712_TSIT_P   CAAGCAGAAGACGGCATACGAGATTCCTCTACctccgctttcctctctatgggcagtcggtgat      B1   N713_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCTTATCGC   ctccgctttcctctctatgggcagtcggtgat      B2   N714_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTACGCTAGctccgctttcctctctatgggcagtcggtgat      B3   N715_ TSIT_P   CAAGCAGAAGACGGCATACGAGATGCCTTTCTctccgctttcctctctatgggcagtcggtgat      B4   N716_ TSIT_P   CAAGCAGAAGACGGCATACGAGATAGGAGCTCctccgctttcctctctatgggcagtcggtgat      B5   N717_ TSIT_P   CAAGCAGAAGACGGCATACGAGATGTCCAGGActccgctttcctctctatgggcagtcggtgat      B6   N718_ TSIT_P    CAAGCAGAAGACGGCATACGAGATCCTACATGctccgctttcctctctatgggcagtcggtgat      B7   N719_ TSIT_P   CAAGCAGAAGACGGCATACGAGATAGAGGTAGctccgctttcctctctatgggcagtcggtgat      B8   N720_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTCTGCCTCctccgctttcctctctatgggcagtcggtgat      B9   N721_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTAGCAGCGctccgctttcctctctatgggcagtcggtgat      B10   N722_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCTCGCAGCctccgctttcctctctatgggcagtcggtgat      B11   N723_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTCTTTGCCctccgctttcctctctatgggcagtcggtgat      B12   N724_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCTACTCCTctccgctttcctctctatgggcagtcggtgat      C1   N725_ TSIT_P   CAAGCAGAAGACGGCATACGAGATGCCTTATCctccgctttcctctctatgggcagtcggtgat      C2   N726_ TSIT_P   CAAGCAGAAGACGGCATACGAGATAGTACGCTctccgctttcctctctatgggcagtcggtgat      C3   N727_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCTGCCTTTctccgctttcctctctatgggcagtcggtgat      C4   N728_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTCAGGAGCctccgctttcctctctatgggcagtcggtgat      C5   N729_ TSIT_P   CAAGCAGAAGACGGCATACGAGATGAGTCCAGctccgctttcctctctatgggcagtcggtgat      C6   N730_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTGCCTACActccgctttcctctctatgggcagtcggtgat      C7   N731_ TSIT_P   CAAGCAGAAGACGGCATACGAGATAGAGAGGTctccgctttcctctctatgggcagtcggtgat      C8   N732_ TSIT_P   CAAGCAGAAGACGGCATACGAGATTCTCTGCCctccgctttcctctctatgggcagtcggtgat      C9   N733_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCGTAGCAGctccgctttcctctctatgggcagtcggtgat      C10   N734_ TSIT_P   CAAGCAGAAGACGGCATACGAGATGCCTCGCActccgctttcctctctatgggcagtcggtgat      C11   N735_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCCTCTTTGctccgctttcctctctatgggcagtcggtgat      C12   N736_ TSIT_P   CAAGCAGAAGACGGCATACGAGATCTCTACTCctccgctttcctctctatgggcagtcggtgat     
 
 
            Plate   well   Name   Sequence      A1   N501_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTAGATCGCccatctcatccctgcgtgtctccgactcag      B1   N502_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCTCTCTATccatctcatccctgcgtgtctccgactcag      C1   N503_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTATCCTCTccatctcatccctgcgtgtctccgactcag      D1   N504_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAGAGTAGAccatctcatccctgcgtgtctccgactcag      E1   N505_TSIT_A   AATGATACGGCGACCACCGAGATCTACACGTAAGGAGccatctcatccctgcgtgtctccgactcag      F1   N506_TSIT_A   AATGATACGGCGACCACCGAGATCTACACACTGCATAccatctcatccctgcgtgtctccgactcag      G1   N507_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAAGGAGTAccatctcatccctgcgtgtctccgactcag      H1   N508_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCTAAGCCTccatctcatccctgcgtgtctccgactcag      A2   N509_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTCGCTAGAccatctcatccctgcgtgtctccgactcag      B2   N510_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCTATCTCTccatctcatccctgcgtgtctccgactcag      C2   N511_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCTCTTATCccatctcatccctgcgtgtctccgactcag      D2   N512_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTAGAAGAGccatctcatccctgcgtgtctccgactcag      E2   N513_TSIT_A   AATGATACGGCGACCACCGAGATCTACACGGAGGTAAccatctcatccctgcgtgtctccgactcag      F2   N514_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCATAACTGccatctcatccctgcgtgtctccgactcag      G2   N515_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAGTAAAGGccatctcatccctgcgtgtctccgactcag      H2   N516_TSIT_A   AATGATACGGCGACCACCGAGATCTACACGCCTCTAAccatctcatccctgcgtgtctccgactcag      A3   N517_TSIT_A   AATGATACGGCGACCACCGAGATCTACACGATCGCTAccatctcatccctgcgtgtctccgactcag       B3   N518_TSIT_A   AATGATACGGCGACCACCGAGATCTACACCTCTATCTccatctcatccctgcgtgtctccgactcag      C3   N519_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTCCTCTTAccatctcatccctgcgtgtctccgactcag      D3   N520_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAGTAGAAGccatctcatccctgcgtgtctccgactcag      E3   N521_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAAGGAGGTccatctcatccctgcgtgtctccgactcag      F3   N522_TSIT_A   AATGATACGGCGACCACCGAGATCTACACTGCATAACccatctcatccctgcgtgtctccgactcag      G3   N523_TSIT_A   AATGATACGGCGACCACCGAGATCTACACGGAGTAAAccatctcatccctgcgtgtctccgactcag      H3   N524_TSIT_A   AATGATACGGCGACCACCGAGATCTACACAAGCCTCTccatctcatccctgcgtgtctccgactcag     
 

TSITs preparation
Make a new plate diluted to 50 µM from each TSIT plate (TSIT_A and TSIT_P) at 100 or 200 µM in 100 µL/TSIT in TE: TSIT_A_50µM and TSIT_P_50µM.
       Ci µM         Cf µM         Vi µL         Vf         V TE            200         50         50         200         150            100         50         100         200         100      

Finally prepare 10 µM plates by mixing 2 by 2 TSIT_A with TSIT_P to obtain 9 plates of different combinations (plates are named TSIT_10µM_1 to TSIT_10µM_9).
As the TSIT_A are arranged columnwise in plate TSIT_A_50µM and TSIT_P are sorted rowise in plate TSIT_P_50µM, distribute 5µL of each column of TSIT_A and each row of TSIT_P in the 9 plates according to the table below.  
       Plate name TSIT_10µM         TSIT_A column         TSIT_P row         Format            TSIT_10µM_1         1         A         96-well PCR Plate            TSIT_10µM_2         1         B         96-well PCR Plate            TSIT_10µM_3         1         C         96-well PCR Plate            TSIT_10µM_4         2         A         96-well PCR Plate            TSIT_10µM_5         2         B         96-well PCR Plate            TSIT_10µM_6         2         C         96-well PCR Plate            TSIT_10µM_7         3         A         96-well PCR Plate            TSIT_10µM_8         3         B         96-well PCR Plate            TSIT_10µM_9         3         C         96-well PCR Plate      

  

17. Summary
SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
4. Important informations before starting PCRs

Please read the GUIDELINES & WARNINGS to set up appropriate genotyping experiments. Make sure your number of samples is a multiple of 96 and the sequencing strategy you have chosen allows to reach 100X read depth for each sample at each locus. Note that replicating some samples can provide interesting information about genotyping quality (we run at least one set of 96 samples in triplicate for each new species or SNP panel we analyze).

Then,
prepare a PCR plate layout,
and determine the TSIT combinations that will be used for each sample/DNA plate. Make sure not to use the same TSIT plate twice in the same sequencing run. Each barcode combination must be unique.

For each PCR, we advise to report those informations in tables, for example:
 

SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
6. Pooling PCR products
Centrifuge each PCR plate before remove sealing foil film.
on ice Prepare one 2mL tube per 96-well PCR plate. Use a multichannel pipette to transfer 20 µL of each PCR product to a tube strip. Combine all contents of the tube strip into a 2 mL microcentrifuge tube. For each 96-well plate, the final volume must reach 1,920 µL.
Store at 4 ˚C until the next step or at -20°C for longer storage.
SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
7. Cleaning-up PCR pool on magnetic beads

  
Room temperature 
Ensure magnetic beads have been placed at room temperature for at least 30 minutes before starting the clean-up step. All steps are run twice to ensure optimal library preparation.
 
Bind target DNA fragment onto magnetic beads
To remove the smallest fragments and primer dimers, use a ratio 0.9/1 (beads/sample).
Transfer 960 µL of PCR products to a new 2 mL tube. Leave the remaining 960 µL at 4 ˚C until the end of the protocol or at -20 ˚C for longer term storage.
Add 864µL of presuspended magnetic beads (0.9x960µL=864µL).
Mix up and down 10 times with the pipette.
Incubate at room temperature for 5 min.
Place the tube in a magnetic rack and wait until all the beads are against the side of the tube. This could take a few minutes. 
Remove the supernatant without removing any bead.

Wash the beads with 85% ethanol
Add 500 µL of 85% ethanol to the beads and incubate at room temperature for 30 s.
Return the tube to the magnetic tube strip.
Remove the supernatant without removing any bead.
Repeat step 1 (second washing step).
Let beads dry on the magnetic rack for 5-15 min at room temperature.

Elute DNA
To elute DNA, remove the tubes from the magnetic rack. 
Add 70 µL of DNase/RNase free water and mix by pipetting up and down 10 times.
Incubate at room temperature for 1-5 min. 
Place the tube to the magnetic rack and wait for the beads to be against the magnetic strip.
Transfer the 70µl of water containing target DNA into a new 1.5 or 2 mL tube.

Bind eluted DNA onto magnetic beads
Add 63µL of presuspend magnetic beads (0.9x70µL=63µL) to the previously eluted DNA.
Mix up and down 10 times.
Incubate at room temperature for 5 min.
Place the tube in a magnetic rack and wait until all the beads are against the side of the tube. This could take a few minutes. 
Remove the supernatant without removing any bead.

Wash the beads with 85% ethanol
Add 180 µL of 85% ethanol to the beads and incubate at room temperature for 30 s.
Return tube to the magnetic tube strip.
Remove the supernatant without removing any bead.
Let beads dry on magnetic rack for 5-15 min at room temperature.

Elute DNA
To elute DNA, remove the tubes from the magnetic rack. 
Add 70 µL of DNase/RNase free water and mix by pipetting up and down 10 times.
Incubate at room temperature for 1-5 min. 
Place the tube to the magnetic rack and wait for the beads to be against the magnetic strip.
Transfer the 70µl of water containing target DNA into a new 1.5 mL tube.
17. Size selction and gel purification (optionnal)
SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
8. PCR2 - Fragment enrichment

PCR mix
=> Keep all reagents, PCR plate on ice or cold cube during all process.
Prepare the PCR mix followong indications described in the table below. Adjust volumes to the number of purified PCR1 product and controls needed. The PCR mix can be used right away or stored on ice or at 4 ˚C for several hours.
Distribute 10 µL of purified PCR1 product (or control) to each well in strip-tubes.
Distribute 45 µL of mix to each well.
Cap each tube.
 
PCR cycles
Program the PCR machine according to the following table :

 

SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
9. Size selection

Option 1: Size selection by electrophoresis in agarose gel

Agarose gel electrophoresis
Important: the electrophoresis comb must afford 55 µL loading volumes.
Prepare a 1.75% agarose gel using 1X TAE and ethidium bromide (EtBr). 
Add directly 55µL of each sample to a well (possible due to the use of the green buffer in previous PCR2 step)
Add 10 µL of the 50 bp ladder to at least one well (adapat to the number of samples or your gel size).
Run the gel at 100V for about 50-75 min to get more than 5 cm migration.

Size selection
Cut out an agarose gel band between 250-300 bp with a scalpel for each sample.
Weigh an empty 2 mL microcentrifuge tube. Then, add the gel slice to the tube and re-weigh.
Note the weigh of the agarose gel slice to adapt the correct volume of NTI buffer in next step.

DNA purification on silica column
Use the MN Nucleospin gel clean-up kit using the gel extraction protocol supplied by the manufacturer.
Prepare the NT3 and add the appropriate volume of 96-100% ethanol.
Adapt the volume of NTI buffer for each sample following : Vol. NTI = 2x agarose weight.
Elute with 2 x 25 µL of buffer NE (50 µL total).
Store at 4 ˚C.
The samples are now ready for the sequencing platform.

  

   
Option 2: Size selection by PippinTM prep

Bring each DNA sample from PCR2 up to 60μl with water.
Combine 60µl of DNA sample with 20μl of loading/marker mix (Marker L).
Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.
Remove 40μl of buffer from two "Sample Well" of the cassette, and load 40μl of sample into each well (2 wells per sample).
For each sample cut out band between 250-300 bp with “Range” option in PippinTM Prep software.
After migration, remove 40µl of sample from the two "Elution Module" of the cassette.





SECTION: Summary
1. Foreword
The protocol that is described hereafter is adapted from Nguyen-Dumont et al. (2013) and Hammet et al. (2019). In this Summary section, potential alternatives to the protocol are mentioned in italic. These alternatives are not described later on (from section 2 onwards).

Protocol aim
The Hi-Plex protocol we describe allows the co-amplification and subsequent sequencing of targeted SNP panels (up to 456 SNPs in our trials), and runs through different steps: the synthesis and preparation of gene specifics primers (GSPs) and adaptors (TSITs); a selective multiplex amplification of the SNP panel in a two-step PCR that allows building amplicons, the pooling of PCR products, a second PCR to increase the number of amplicon copies, interrupted with clean-up steps; quality control of the pooled libraries and their sequencing with custom sequencing primers. The sequencing-specific sequences included in the library constructions allow for both Illumina or Ion Torrent sequencing but we only tested and thereafter show the protocol for Illumina Miseq sequencing.

Gene specific primers (GSPs) 
GSPs are forward and reverse oligos designed from target fragments of 145bp for each SNP. Each resulting Gene Specific Primer (GSP) is built from a target specific sequence and a common heel used to add individual index and sequencing oligos (TSIT).

Adaptors (TSITs)
TSITs are adaptors that consist of a complementary sequence to the GSP common heel, a barcode, and a specific sequence for Illumina sequencing (P5/P7) for a total of about 60 bp for each TSIT A and TSIT P. The use of a combination of this 2 barcodes allows the specific identification of the DNA samples.

Library preparation
The first PCR uses a high fidelity hot start taq polymerase and is made of two phases. In the first phase, a pool of all GSPs (50 µM) is used, to which TSIT adaptors are added for the second phase. The first PCR phase starts with 8 cycles with 2 successive hybridization temperatures (TM) at 58˚C and 60˚C, followed by 4 cycles at 58˚C. The second phase includes 4 cycles with a TM at 66˚C. The reaction is stopped by adding 100 mM EDTA at 72 ˚C and putting on ice.

The amplifications are pooled (the pooling unit is a 96-well plate) and purified using magnetic beads. Then, the second PCR is carried out using a high fidelity taq polymerase and the two Illumina's primers P5 and P7 at TM = 58 ˚C. The amplified fragments (250-300 bp) are size selected by electrophoresis from an agarose gel colored with EtBr followed by extraction with a commercially available silica column or from an automatically size-selection system such as the PipinnTM prep. 

The use of EtBr is essential if size selection from agarose gel is performed. SybrSafe reagent, for example, generated a lot of non-specific amplicons. This suggests that SybrSafe does not allow for clean separation of migrated fragments on agarose gels.

    
Quality controls and sequencing
Each library is checked on a Fragment Analyzer™ to estimate the purity of the amplification products. Library concentration is then measured by KAPA qPCR on a LightCycler according to the manufacter protocol. All libraries are finally pooled together in equimolar quantities for sequencing performed on a Miseq platform (Illumina).

   
SECTION: PCR Preparation and Clean-Up for SNP genotyping using Hi-Plex
5. PCR1 - SNP amplification step

on ice PCR mix for one 96-well PCR plate
=> Keep all reagents, PCR plate on ice or cold cube during all process.
Prepare the PCR mix following the table below. The PCR mix can be used right away or stored on ice or at 4 ˚C for several hours.
Distribute 2 µL of DNA (or control) into each well – make sure to design a plate layout for the samples.
Distribute 21 µL of mix to each well.
Cover the PCR plate with foil sealing films and keep on ice until PCR running.

 

PCR cycles

 
To limit aspecific amplification, the PCR program requires a pre-heating step at 98 ˚C. Start the PCR program without inserting the plate. Store the plate on ice until the heating block on the machine has reached 98 ˚C. Then, insert the plate.
At the 1st step at 72°C forever, stop the run and open the thermocycler. Keep the PCR plate in the PCR machine at 72 ˚C.
Take the appropriate TSIT_10µM plates stored at 4 ˚C, centrifuge the plate, and carefully remove the sealing film.
Carefully remove the PCR plate sealing film.
Add 2.5 µL of each TSIT_10µM to each column of the PCR plate with a multichannel pipette. Mix by carefully pipetting up and down. Change tips for each column.
Close the PCR and TSIT_10µM plates with new sealing films. Restart the PCR program for the following steps.
At the final step at 72°C forever, stop the run and open the thermocycler. Keep the PCR plate in the PCR machine at 72 ˚C.
Take the EDTA 100mM in tube strips stored at 4°C, centrifuge the strip and carefully open up the caps.
Carefully remove the PCR plate sealing film.
Add 2.5 µL of EDTA 100mM to each column of the PCR plate with a multichannel pipette. Mix by carefully pipetting up and down. Change tips for each column.
Seal the plate with a new storage sealing foil film and put on ice immediately.
on ice Plates are placed directly on ice and stored at 4°C until the next steps.

SECTION: Quality and quantity control of libraries
10. Check the purity of each library on a Fragment Analyzer™
Use Invitrogen™ Qubit™ Fluorometer or equivalent to estimate DNA concentrations for each library
Prepare a 50pg/µL to 5ng/µL dilution of each library.
Then, follow the instructions from the manufacter.

  
  
  

SECTION: Short information for sequencing Hi-Plex libraries on the ILLUMNA Miseq system
13. Custom primers
This method uses custom primers which are in positions 12, 13, and 14 of the MiSeq reagent cartridge. For more information or for using other ILLUMINA System, refer to "Custom Primers Guide" on ILLUMiNA website.
       Primers         Séquence 5’-3’         Emplacement pour le run         Volume            TSIT_Read1         ccatctcatccctgcgtgtctccgactcag         Réservoir 12         4µL/run à   100µM             TSIT_i7_read         aatcaccgactgcccatagagaggaaagcggag         Réservoir 13         4µL/run à   100µM            TSIT_Read2         ctccgctttcctctctatgggcagtcggtgatt         Réservoir 14         4µL/run à   100µM      

SECTION: Short information for sequencing Hi-Plex libraries on the ILLUMNA Miseq system
12. Informations for samplesheets
Report the corresponding "barcod P complement" and "barcod A" according to TSIT plate added at PCR1 step.
 
        Plate   well   TSIT_1   TSIT_1   TSIT_2   TSIT_2   TSIT_3   TSIT_3   TSIT_4   TSIT_4   TSIT_5   TSIT_5   TSIT_6   TSIT_6   TSIT_7   TSIT_7   TSIT_8   TSIT_8   TSIT_9   TSIT_9         P complement   Barcod A   P complement   Barcod A   P complement   Barcod A   P complement   Barcod A   P complement   Barcod A   P complement   BarcodA   P complement   BarcodA   P complement   BarcodA   P complement   BarcodA      A1   TAAGGCGA   TAGATCGC   GCGATAAG   TAGATCGC   GATAAGGC   TAGATCGC   TAAGGCGA   TCGCTAGA   GCGATAAG   TCGCTAGA   GATAAGGC   TCGCTAGA   TAAGGCGA   GATCGCTA   GCGATAAG   GATCGCTA   GATAAGGC   GATCGCTA      B1   TAAGGCGA   CTCTCTAT   GCGATAAG   CTCTCTAT   GATAAGGC   CTCTCTAT   TAAGGCGA   CTATCTCT   GCGATAAG   CTATCTCT   GATAAGGC   CTATCTCT   TAAGGCGA   CTCTATCT   GCGATAAG   CTCTATCT   GATAAGGC   CTCTATCT      C1   TAAGGCGA   TATCCTCT   GCGATAAG   TATCCTCT   GATAAGGC   TATCCTCT   TAAGGCGA   CTCTTATC   GCGATAAG   CTCTTATC   GATAAGGC   CTCTTATC   TAAGGCGA   TCCTCTTA   GCGATAAG   TCCTCTTA   GATAAGGC   TCCTCTTA      D1   TAAGGCGA   AGAGTAGA   GCGATAAG   AGAGTAGA   GATAAGGC   AGAGTAGA   TAAGGCGA   TAGAAGAG   GCGATAAG   TAGAAGAG   GATAAGGC   TAGAAGAG   TAAGGCGA   AGTAGAAG   GCGATAAG   AGTAGAAG   GATAAGGC   AGTAGAAG      E1   TAAGGCGA   GTAAGGAG   GCGATAAG   GTAAGGAG   GATAAGGC   GTAAGGAG   TAAGGCGA   GGAGGTAA   GCGATAAG   GGAGGTAA   GATAAGGC   GGAGGTAA   TAAGGCGA   AAGGAGGT   GCGATAAG   AAGGAGGT   GATAAGGC   AAGGAGGT      F1   TAAGGCGA   ACTGCATA   GCGATAAG   ACTGCATA   GATAAGGC   ACTGCATA   TAAGGCGA   CATAACTG   GCGATAAG   CATAACTG   GATAAGGC   CATAACTG   TAAGGCGA   TGCATAAC   GCGATAAG   TGCATAAC   GATAAGGC   TGCATAAC      G1   TAAGGCGA   AAGGAGTA   GCGATAAG   AAGGAGTA   GATAAGGC   AAGGAGTA   TAAGGCGA   AGTAAAGG   GCGATAAG   AGTAAAGG   GATAAGGC   AGTAAAGG   TAAGGCGA   GGAGTAAA   GCGATAAG   GGAGTAAA   GATAAGGC   GGAGTAAA      H1   TAAGGCGA   CTAAGCCT   GCGATAAG   CTAAGCCT   GATAAGGC   CTAAGCCT   TAAGGCGA   GCCTCTAA   GCGATAAG   GCCTCTAA   GATAAGGC   GCCTCTAA   TAAGGCGA   AAGCCTCT   GCGATAAG   AAGCCTCT   GATAAGGC   AAGCCTCT      A2   CGTACTAG   TAGATCGC   CTAGCGTA   TAGATCGC   AGCGTACT   TAGATCGC   CGTACTAG   TCGCTAGA   CTAGCGTA   TCGCTAGA   AGCGTACT   TCGCTAGA   CGTACTAG   GATCGCTA   CTAGCGTA   GATCGCTA   AGCGTACT   GATCGCTA      B2   CGTACTAG   CTCTCTAT   CTAGCGTA   CTCTCTAT   AGCGTACT   CTCTCTAT   CGTACTAG   CTATCTCT   CTAGCGTA   CTATCTCT   AGCGTACT   CTATCTCT   CGTACTAG   CTCTATCT   CTAGCGTA   CTCTATCT   AGCGTACT   CTCTATCT      C2   CGTACTAG   TATCCTCT   CTAGCGTA   TATCCTCT   AGCGTACT   TATCCTCT   CGTACTAG   CTCTTATC   CTAGCGTA   CTCTTATC   AGCGTACT   CTCTTATC   CGTACTAG   TCCTCTTA   CTAGCGTA   TCCTCTTA   AGCGTACT   TCCTCTTA      D2   CGTACTAG   AGAGTAGA   CTAGCGTA   AGAGTAGA   AGCGTACT   AGAGTAGA   CGTACTAG   TAGAAGAG   CTAGCGTA   TAGAAGAG   AGCGTACT   TAGAAGAG   CGTACTAG   AGTAGAAG   CTAGCGTA   AGTAGAAG   AGCGTACT   AGTAGAAG      E2   CGTACTAG   GTAAGGAG   CTAGCGTA   GTAAGGAG   AGCGTACT   GTAAGGAG   CGTACTAG   GGAGGTAA   CTAGCGTA   GGAGGTAA   AGCGTACT   GGAGGTAA   CGTACTAG   AAGGAGGT   CTAGCGTA   AAGGAGGT   AGCGTACT   AAGGAGGT      F2   CGTACTAG   ACTGCATA   CTAGCGTA   ACTGCATA   AGCGTACT   ACTGCATA   CGTACTAG   CATAACTG   CTAGCGTA   CATAACTG   AGCGTACT   CATAACTG   CGTACTAG   TGCATAAC   CTAGCGTA   TGCATAAC   AGCGTACT   TGCATAAC      G2   CGTACTAG   AAGGAGTA   CTAGCGTA   AAGGAGTA   AGCGTACT   AAGGAGTA   CGTACTAG   AGTAAAGG   CTAGCGTA   AGTAAAGG   AGCGTACT   AGTAAAGG   CGTACTAG   GGAGTAAA   CTAGCGTA   GGAGTAAA   AGCGTACT   GGAGTAAA      H2   CGTACTAG   CTAAGCCT   CTAGCGTA   CTAAGCCT   AGCGTACT   CTAAGCCT   CGTACTAG   GCCTCTAA   CTAGCGTA   GCCTCTAA   AGCGTACT   GCCTCTAA   CGTACTAG   AAGCCTCT   CTAGCGTA   AAGCCTCT   AGCGTACT   AAGCCTCT      A3   AGGCAGAA   TAGATCGC   AGAAAGGC   TAGATCGC   AAAGGCAG   TAGATCGC   AGGCAGAA   TCGCTAGA   AGAAAGGC   TCGCTAGA   AAAGGCAG   TCGCTAGA   AGGCAGAA   GATCGCTA   AGAAAGGC   GATCGCTA   AAAGGCAG   GATCGCTA      B3   AGGCAGAA   CTCTCTAT   AGAAAGGC   CTCTCTAT   AAAGGCAG   CTCTCTAT   AGGCAGAA   CTATCTCT   AGAAAGGC   CTATCTCT   AAAGGCAG   CTATCTCT   AGGCAGAA   CTCTATCT   AGAAAGGC   CTCTATCT   AAAGGCAG   CTCTATCT      C3   AGGCAGAA   TATCCTCT   AGAAAGGC   TATCCTCT   AAAGGCAG   TATCCTCT   AGGCAGAA   CTCTTATC   AGAAAGGC   CTCTTATC   AAAGGCAG   CTCTTATC   AGGCAGAA   TCCTCTTA   AGAAAGGC   TCCTCTTA   AAAGGCAG   TCCTCTTA      D3   AGGCAGAA   AGAGTAGA   AGAAAGGC   AGAGTAGA   AAAGGCAG   AGAGTAGA   AGGCAGAA   TAGAAGAG   AGAAAGGC   TAGAAGAG   AAAGGCAG   TAGAAGAG   AGGCAGAA   AGTAGAAG   AGAAAGGC   AGTAGAAG   AAAGGCAG   AGTAGAAG      E3   AGGCAGAA   GTAAGGAG   AGAAAGGC   GTAAGGAG   AAAGGCAG   GTAAGGAG   AGGCAGAA   GGAGGTAA   AGAAAGGC   GGAGGTAA   AAAGGCAG   GGAGGTAA   AGGCAGAA   AAGGAGGT   AGAAAGGC   AAGGAGGT   AAAGGCAG   AAGGAGGT      F3   AGGCAGAA   ACTGCATA   AGAAAGGC   ACTGCATA   AAAGGCAG   ACTGCATA   AGGCAGAA   CATAACTG   AGAAAGGC   CATAACTG   AAAGGCAG   CATAACTG   AGGCAGAA   TGCATAAC   AGAAAGGC   TGCATAAC   AAAGGCAG   TGCATAAC      G3   AGGCAGAA   AAGGAGTA   AGAAAGGC   AAGGAGTA   AAAGGCAG   AAGGAGTA   AGGCAGAA   AGTAAAGG   AGAAAGGC   AGTAAAGG   AAAGGCAG   AGTAAAGG   AGGCAGAA   GGAGTAAA   AGAAAGGC   GGAGTAAA   AAAGGCAG   GGAGTAAA      H3   AGGCAGAA   CTAAGCCT   AGAAAGGC   CTAAGCCT   AAAGGCAG   CTAAGCCT   AGGCAGAA   GCCTCTAA   AGAAAGGC   GCCTCTAA   AAAGGCAG   GCCTCTAA   AGGCAGAA   AAGCCTCT   AGAAAGGC   AAGCCTCT   AAAGGCAG   AAGCCTCT      A4   TCCTGAGC   TAGATCGC   GAGCTCCT   TAGATCGC   GCTCCTGA   TAGATCGC   TCCTGAGC   TCGCTAGA   GAGCTCCT   TCGCTAGA   GCTCCTGA   TCGCTAGA   TCCTGAGC   GATCGCTA   GAGCTCCT   GATCGCTA   GCTCCTGA   GATCGCTA      B4   TCCTGAGC   CTCTCTAT   GAGCTCCT   CTCTCTAT   GCTCCTGA   CTCTCTAT   TCCTGAGC   CTATCTCT   GAGCTCCT   CTATCTCT   GCTCCTGA   CTATCTCT   TCCTGAGC   CTCTATCT   GAGCTCCT   CTCTATCT   GCTCCTGA   CTCTATCT      C4   TCCTGAGC   TATCCTCT   GAGCTCCT   TATCCTCT   GCTCCTGA   TATCCTCT   TCCTGAGC   CTCTTATC   GAGCTCCT   CTCTTATC   GCTCCTGA   CTCTTATC   TCCTGAGC   TCCTCTTA   GAGCTCCT   TCCTCTTA   GCTCCTGA   TCCTCTTA      D4   TCCTGAGC   AGAGTAGA   GAGCTCCT   AGAGTAGA   GCTCCTGA   AGAGTAGA   TCCTGAGC   TAGAAGAG   GAGCTCCT   TAGAAGAG   GCTCCTGA   TAGAAGAG   TCCTGAGC   AGTAGAAG   GAGCTCCT   AGTAGAAG   GCTCCTGA   AGTAGAAG      E4   TCCTGAGC   GTAAGGAG   GAGCTCCT   GTAAGGAG   GCTCCTGA   GTAAGGAG   TCCTGAGC   GGAGGTAA   GAGCTCCT   GGAGGTAA   GCTCCTGA   GGAGGTAA   TCCTGAGC   AAGGAGGT   GAGCTCCT   AAGGAGGT   GCTCCTGA   AAGGAGGT      F4   TCCTGAGC   ACTGCATA   GAGCTCCT   ACTGCATA   GCTCCTGA   ACTGCATA   TCCTGAGC   CATAACTG   GAGCTCCT   CATAACTG   GCTCCTGA   CATAACTG   TCCTGAGC   TGCATAAC   GAGCTCCT   TGCATAAC   GCTCCTGA   TGCATAAC      G4   TCCTGAGC   AAGGAGTA   GAGCTCCT   AAGGAGTA   GCTCCTGA   AAGGAGTA   TCCTGAGC   AGTAAAGG   GAGCTCCT   AGTAAAGG   GCTCCTGA   AGTAAAGG   TCCTGAGC   GGAGTAAA   GAGCTCCT   GGAGTAAA   GCTCCTGA   GGAGTAAA      H4   TCCTGAGC   CTAAGCCT   GAGCTCCT   CTAAGCCT   GCTCCTGA   CTAAGCCT   TCCTGAGC   GCCTCTAA   GAGCTCCT   GCCTCTAA   GCTCCTGA   GCCTCTAA   TCCTGAGC   AAGCCTCT   GAGCTCCT   AAGCCTCT   GCTCCTGA   AAGCCTCT      A5   GGACTCCT   TAGATCGC   TCCTGGAC   TAGATCGC   CTGGACTC   TAGATCGC   GGACTCCT   TCGCTAGA   TCCTGGAC   TCGCTAGA   CTGGACTC   TCGCTAGA   GGACTCCT   GATCGCTA   TCCTGGAC   GATCGCTA   CTGGACTC   GATCGCTA      B5   GGACTCCT   CTCTCTAT   TCCTGGAC   CTCTCTAT   CTGGACTC   CTCTCTAT   GGACTCCT   CTATCTCT   TCCTGGAC   CTATCTCT   CTGGACTC   CTATCTCT   GGACTCCT   CTCTATCT   TCCTGGAC   CTCTATCT   CTGGACTC   CTCTATCT      C5   GGACTCCT   TATCCTCT   TCCTGGAC   TATCCTCT   CTGGACTC   TATCCTCT   GGACTCCT   CTCTTATC   TCCTGGAC   CTCTTATC   CTGGACTC   CTCTTATC   GGACTCCT   TCCTCTTA   TCCTGGAC   TCCTCTTA   CTGGACTC   TCCTCTTA      D5   GGACTCCT   AGAGTAGA   TCCTGGAC   AGAGTAGA   CTGGACTC   AGAGTAGA   GGACTCCT   TAGAAGAG   TCCTGGAC   TAGAAGAG   CTGGACTC   TAGAAGAG   GGACTCCT   AGTAGAAG   TCCTGGAC   AGTAGAAG   CTGGACTC   AGTAGAAG      E5   GGACTCCT   GTAAGGAG   TCCTGGAC   GTAAGGAG   CTGGACTC   GTAAGGAG   GGACTCCT   GGAGGTAA   TCCTGGAC   GGAGGTAA   CTGGACTC   GGAGGTAA   GGACTCCT   AAGGAGGT   TCCTGGAC   AAGGAGGT   CTGGACTC   AAGGAGGT      F5   GGACTCCT   ACTGCATA   TCCTGGAC   ACTGCATA   CTGGACTC   ACTGCATA   GGACTCCT   CATAACTG   TCCTGGAC   CATAACTG   CTGGACTC   CATAACTG   GGACTCCT   TGCATAAC   TCCTGGAC   TGCATAAC   CTGGACTC   TGCATAAC      G5   GGACTCCT   AAGGAGTA   TCCTGGAC   AAGGAGTA   CTGGACTC   AAGGAGTA   GGACTCCT   AGTAAAGG   TCCTGGAC   AGTAAAGG   CTGGACTC   AGTAAAGG   GGACTCCT   GGAGTAAA   TCCTGGAC   GGAGTAAA   CTGGACTC   GGAGTAAA      H5   GGACTCCT   CTAAGCCT   TCCTGGAC   CTAAGCCT   CTGGACTC   CTAAGCCT   GGACTCCT   GCCTCTAA   TCCTGGAC   GCCTCTAA   CTGGACTC   GCCTCTAA   GGACTCCT   AAGCCTCT   TCCTGGAC   AAGCCTCT   CTGGACTC   AAGCCTCT      A6   TAGGCATG   TAGATCGC   CATGTAGG   TAGATCGC   TGTAGGCA   TAGATCGC   TAGGCATG   TCGCTAGA   CATGTAGG   TCGCTAGA   TGTAGGCA   TCGCTAGA   TAGGCATG   GATCGCTA   CATGTAGG   GATCGCTA   TGTAGGCA   GATCGCTA      B6   TAGGCATG   CTCTCTAT   CATGTAGG   CTCTCTAT   TGTAGGCA   CTCTCTAT   TAGGCATG   CTATCTCT   CATGTAGG   CTATCTCT   TGTAGGCA   CTATCTCT   TAGGCATG   CTCTATCT   CATGTAGG   CTCTATCT   TGTAGGCA   CTCTATCT      C6   TAGGCATG   TATCCTCT   CATGTAGG   TATCCTCT   TGTAGGCA   TATCCTCT   TAGGCATG   CTCTTATC   CATGTAGG   CTCTTATC   TGTAGGCA   CTCTTATC   TAGGCATG   TCCTCTTA   CATGTAGG   TCCTCTTA   TGTAGGCA   TCCTCTTA      D6   TAGGCATG   AGAGTAGA   CATGTAGG   AGAGTAGA   TGTAGGCA   AGAGTAGA   TAGGCATG   TAGAAGAG   CATGTAGG   TAGAAGAG   TGTAGGCA   TAGAAGAG   TAGGCATG   AGTAGAAG   CATGTAGG   AGTAGAAG   TGTAGGCA   AGTAGAAG      E6   TAGGCATG   GTAAGGAG   CATGTAGG   GTAAGGAG   TGTAGGCA   GTAAGGAG   TAGGCATG   GGAGGTAA   CATGTAGG   GGAGGTAA   TGTAGGCA   GGAGGTAA   TAGGCATG   AAGGAGGT   CATGTAGG   AAGGAGGT   TGTAGGCA   AAGGAGGT      F6   TAGGCATG   ACTGCATA   CATGTAGG   ACTGCATA   TGTAGGCA   ACTGCATA   TAGGCATG   CATAACTG   CATGTAGG   CATAACTG   TGTAGGCA   CATAACTG   TAGGCATG   TGCATAAC   CATGTAGG   TGCATAAC   TGTAGGCA   TGCATAAC      G6   TAGGCATG   AAGGAGTA   CATGTAGG   AAGGAGTA   TGTAGGCA   AAGGAGTA   TAGGCATG   AGTAAAGG   CATGTAGG   AGTAAAGG   TGTAGGCA   AGTAAAGG   TAGGCATG   GGAGTAAA   CATGTAGG   GGAGTAAA   TGTAGGCA   GGAGTAAA      H6   TAGGCATG   CTAAGCCT   CATGTAGG   CTAAGCCT   TGTAGGCA   CTAAGCCT   TAGGCATG   GCCTCTAA   CATGTAGG   GCCTCTAA   TGTAGGCA   GCCTCTAA   TAGGCATG   AAGCCTCT   CATGTAGG   AAGCCTCT   TGTAGGCA   AAGCCTCT      A7   CTCTCTAC   TAGATCGC   CTACCTCT   TAGATCGC   ACCTCTCT   TAGATCGC   CTCTCTAC   TCGCTAGA   CTACCTCT   TCGCTAGA   ACCTCTCT   TCGCTAGA   CTCTCTAC   GATCGCTA   CTACCTCT   GATCGCTA   ACCTCTCT   GATCGCTA      B7   CTCTCTAC   CTCTCTAT   CTACCTCT   CTCTCTAT   ACCTCTCT   CTCTCTAT   CTCTCTAC   CTATCTCT   CTACCTCT   CTATCTCT   ACCTCTCT   CTATCTCT   CTCTCTAC   CTCTATCT   CTACCTCT   CTCTATCT   ACCTCTCT   CTCTATCT      C7   CTCTCTAC   TATCCTCT   CTACCTCT   TATCCTCT   ACCTCTCT   TATCCTCT   CTCTCTAC   CTCTTATC   CTACCTCT   CTCTTATC   ACCTCTCT   CTCTTATC   CTCTCTAC   TCCTCTTA   CTACCTCT   TCCTCTTA   ACCTCTCT   TCCTCTTA      D7   CTCTCTAC   AGAGTAGA   CTACCTCT   AGAGTAGA   ACCTCTCT   AGAGTAGA   CTCTCTAC   TAGAAGAG   CTACCTCT   TAGAAGAG   ACCTCTCT   TAGAAGAG   CTCTCTAC   AGTAGAAG   CTACCTCT   AGTAGAAG   ACCTCTCT   AGTAGAAG      E7   CTCTCTAC   GTAAGGAG   CTACCTCT   GTAAGGAG   ACCTCTCT   GTAAGGAG   CTCTCTAC   GGAGGTAA   CTACCTCT   GGAGGTAA   ACCTCTCT   GGAGGTAA   CTCTCTAC   AAGGAGGT   CTACCTCT   AAGGAGGT   ACCTCTCT   AAGGAGGT      F7   CTCTCTAC   ACTGCATA   CTACCTCT   ACTGCATA   ACCTCTCT   ACTGCATA   CTCTCTAC   CATAACTG   CTACCTCT   CATAACTG   ACCTCTCT   CATAACTG   CTCTCTAC   TGCATAAC   CTACCTCT   TGCATAAC   ACCTCTCT   TGCATAAC      G7   CTCTCTAC   AAGGAGTA   CTACCTCT   AAGGAGTA   ACCTCTCT   AAGGAGTA   CTCTCTAC   AGTAAAGG   CTACCTCT   AGTAAAGG   ACCTCTCT   AGTAAAGG   CTCTCTAC   GGAGTAAA   CTACCTCT   GGAGTAAA   ACCTCTCT   GGAGTAAA      H7   CTCTCTAC   CTAAGCCT   CTACCTCT   CTAAGCCT   ACCTCTCT   CTAAGCCT   CTCTCTAC   GCCTCTAA   CTACCTCT   GCCTCTAA   ACCTCTCT   GCCTCTAA   CTCTCTAC   AAGCCTCT   CTACCTCT   AAGCCTCT   ACCTCTCT   AAGCCTCT      A8   CAGAGAGG   TAGATCGC   GAGGCAGA   TAGATCGC   GGCAGAGA   TAGATCGC   CAGAGAGG   TCGCTAGA   GAGGCAGA   TCGCTAGA   GGCAGAGA   TCGCTAGA   CAGAGAGG   GATCGCTA   GAGGCAGA   GATCGCTA   GGCAGAGA   GATCGCTA      B8   CAGAGAGG   CTCTCTAT   GAGGCAGA   CTCTCTAT   GGCAGAGA   CTCTCTAT   CAGAGAGG   CTATCTCT   GAGGCAGA   CTATCTCT   GGCAGAGA   CTATCTCT   CAGAGAGG   CTCTATCT   GAGGCAGA   CTCTATCT   GGCAGAGA   CTCTATCT      C8   CAGAGAGG   TATCCTCT   GAGGCAGA   TATCCTCT   GGCAGAGA   TATCCTCT   CAGAGAGG   CTCTTATC   GAGGCAGA   CTCTTATC   GGCAGAGA   CTCTTATC   CAGAGAGG   TCCTCTTA   GAGGCAGA   TCCTCTTA   GGCAGAGA   TCCTCTTA      D8   CAGAGAGG   AGAGTAGA   GAGGCAGA   AGAGTAGA   GGCAGAGA   AGAGTAGA   CAGAGAGG   TAGAAGAG   GAGGCAGA   TAGAAGAG   GGCAGAGA   TAGAAGAG   CAGAGAGG   AGTAGAAG   GAGGCAGA   AGTAGAAG   GGCAGAGA   AGTAGAAG      E8   CAGAGAGG   GTAAGGAG   GAGGCAGA   GTAAGGAG   GGCAGAGA   GTAAGGAG   CAGAGAGG   GGAGGTAA   GAGGCAGA   GGAGGTAA   GGCAGAGA   GGAGGTAA   CAGAGAGG   AAGGAGGT   GAGGCAGA   AAGGAGGT   GGCAGAGA   AAGGAGGT      F8   CAGAGAGG   ACTGCATA   GAGGCAGA   ACTGCATA   GGCAGAGA   ACTGCATA   CAGAGAGG   CATAACTG   GAGGCAGA   CATAACTG   GGCAGAGA   CATAACTG   CAGAGAGG   TGCATAAC   GAGGCAGA   TGCATAAC   GGCAGAGA   TGCATAAC      G8   CAGAGAGG   AAGGAGTA   GAGGCAGA   AAGGAGTA   GGCAGAGA   AAGGAGTA   CAGAGAGG   AGTAAAGG   GAGGCAGA   AGTAAAGG   GGCAGAGA   AGTAAAGG   CAGAGAGG   GGAGTAAA   GAGGCAGA   GGAGTAAA   GGCAGAGA   GGAGTAAA      H8   CAGAGAGG   CTAAGCCT   GAGGCAGA   CTAAGCCT   GGCAGAGA   CTAAGCCT   CAGAGAGG   GCCTCTAA   GAGGCAGA   GCCTCTAA   GGCAGAGA   GCCTCTAA   CAGAGAGG   AAGCCTCT   GAGGCAGA   AAGCCTCT   GGCAGAGA   AAGCCTCT      A9   GCTACGCT   TAGATCGC   CGCTGCTA   TAGATCGC   CTGCTACG   TAGATCGC   GCTACGCT   TCGCTAGA   CGCTGCTA   TCGCTAGA   CTGCTACG   TCGCTAGA   GCTACGCT   GATCGCTA   CGCTGCTA   GATCGCTA   CTGCTACG   GATCGCTA      B9   GCTACGCT   CTCTCTAT   CGCTGCTA   CTCTCTAT   CTGCTACG   CTCTCTAT   GCTACGCT   CTATCTCT   CGCTGCTA   CTATCTCT   CTGCTACG   CTATCTCT   GCTACGCT   CTCTATCT   CGCTGCTA   CTCTATCT   CTGCTACG   CTCTATCT      C9   GCTACGCT   TATCCTCT   CGCTGCTA   TATCCTCT   CTGCTACG   TATCCTCT   GCTACGCT   CTCTTATC   CGCTGCTA   CTCTTATC   CTGCTACG   CTCTTATC   GCTACGCT   TCCTCTTA   CGCTGCTA   TCCTCTTA   CTGCTACG   TCCTCTTA      D9   GCTACGCT   AGAGTAGA   CGCTGCTA   AGAGTAGA   CTGCTACG   AGAGTAGA   GCTACGCT   TAGAAGAG   CGCTGCTA   TAGAAGAG   CTGCTACG   TAGAAGAG   GCTACGCT   AGTAGAAG   CGCTGCTA   AGTAGAAG   CTGCTACG   AGTAGAAG      E9   GCTACGCT   GTAAGGAG   CGCTGCTA   GTAAGGAG   CTGCTACG   GTAAGGAG   GCTACGCT   GGAGGTAA   CGCTGCTA   GGAGGTAA   CTGCTACG   GGAGGTAA   GCTACGCT   AAGGAGGT   CGCTGCTA   AAGGAGGT   CTGCTACG   AAGGAGGT      F9   GCTACGCT   ACTGCATA   CGCTGCTA   ACTGCATA   CTGCTACG   ACTGCATA   GCTACGCT   CATAACTG   CGCTGCTA   CATAACTG   CTGCTACG   CATAACTG   GCTACGCT   TGCATAAC   CGCTGCTA   TGCATAAC   CTGCTACG   TGCATAAC      G9   GCTACGCT   AAGGAGTA   CGCTGCTA   AAGGAGTA   CTGCTACG   AAGGAGTA   GCTACGCT   AGTAAAGG   CGCTGCTA   AGTAAAGG   CTGCTACG   AGTAAAGG   GCTACGCT   GGAGTAAA   CGCTGCTA   GGAGTAAA   CTGCTACG   GGAGTAAA      H9   GCTACGCT   CTAAGCCT   CGCTGCTA   CTAAGCCT   CTGCTACG   CTAAGCCT   GCTACGCT   GCCTCTAA   CGCTGCTA   GCCTCTAA   CTGCTACG   GCCTCTAA   GCTACGCT   AAGCCTCT   CGCTGCTA   AAGCCTCT   CTGCTACG   AAGCCTCT      A10   CGAGGCTG   TAGATCGC   GCTGCGAG   TAGATCGC   TGCGAGGC   TAGATCGC   CGAGGCTG   TCGCTAGA   GCTGCGAG   TCGCTAGA   TGCGAGGC   TCGCTAGA   CGAGGCTG   GATCGCTA   GCTGCGAG   GATCGCTA   TGCGAGGC   GATCGCTA      B10   CGAGGCTG   CTCTCTAT   GCTGCGAG   CTCTCTAT   TGCGAGGC   CTCTCTAT   CGAGGCTG   CTATCTCT   GCTGCGAG   CTATCTCT   TGCGAGGC   CTATCTCT   CGAGGCTG   CTCTATCT   GCTGCGAG   CTCTATCT   TGCGAGGC   CTCTATCT      C10   CGAGGCTG   TATCCTCT   GCTGCGAG   TATCCTCT   TGCGAGGC   TATCCTCT   CGAGGCTG   CTCTTATC   GCTGCGAG   CTCTTATC   TGCGAGGC   CTCTTATC   CGAGGCTG   TCCTCTTA   GCTGCGAG   TCCTCTTA   TGCGAGGC   TCCTCTTA      D10   CGAGGCTG   AGAGTAGA   GCTGCGAG   AGAGTAGA   TGCGAGGC   AGAGTAGA   CGAGGCTG   TAGAAGAG   GCTGCGAG   TAGAAGAG   TGCGAGGC   TAGAAGAG   CGAGGCTG   AGTAGAAG   GCTGCGAG   AGTAGAAG   TGCGAGGC   AGTAGAAG      E10   CGAGGCTG   GTAAGGAG   GCTGCGAG   GTAAGGAG   TGCGAGGC   GTAAGGAG   CGAGGCTG   GGAGGTAA   GCTGCGAG   GGAGGTAA   TGCGAGGC   GGAGGTAA   CGAGGCTG   AAGGAGGT   GCTGCGAG   AAGGAGGT   TGCGAGGC   AAGGAGGT      F10   CGAGGCTG   ACTGCATA   GCTGCGAG   ACTGCATA   TGCGAGGC   ACTGCATA   CGAGGCTG   CATAACTG   GCTGCGAG   CATAACTG   TGCGAGGC   CATAACTG   CGAGGCTG   TGCATAAC   GCTGCGAG   TGCATAAC   TGCGAGGC   TGCATAAC      G10   CGAGGCTG   AAGGAGTA   GCTGCGAG   AAGGAGTA   TGCGAGGC   AAGGAGTA   CGAGGCTG   AGTAAAGG   GCTGCGAG   AGTAAAGG   TGCGAGGC   AGTAAAGG   CGAGGCTG   GGAGTAAA   GCTGCGAG   GGAGTAAA   TGCGAGGC   GGAGTAAA      H10   CGAGGCTG   CTAAGCCT   GCTGCGAG   CTAAGCCT   TGCGAGGC   CTAAGCCT   CGAGGCTG   GCCTCTAA   GCTGCGAG   GCCTCTAA   TGCGAGGC   GCCTCTAA   CGAGGCTG   AAGCCTCT   GCTGCGAG   AAGCCTCT   TGCGAGGC   AAGCCTCT      A11   AAGAGGCA   TAGATCGC   GGCAAAGA   TAGATCGC   CAAAGAGG   TAGATCGC   AAGAGGCA   TCGCTAGA   GGCAAAGA   TCGCTAGA   CAAAGAGG   TCGCTAGA   AAGAGGCA   GATCGCTA   GGCAAAGA   GATCGCTA   CAAAGAGG   GATCGCTA      B11   AAGAGGCA   CTCTCTAT   GGCAAAGA   CTCTCTAT   CAAAGAGG   CTCTCTAT   AAGAGGCA   CTATCTCT   GGCAAAGA   CTATCTCT   CAAAGAGG   CTATCTCT   AAGAGGCA   CTCTATCT   GGCAAAGA   CTCTATCT   CAAAGAGG   CTCTATCT      C11   AAGAGGCA   TATCCTCT   GGCAAAGA   TATCCTCT   CAAAGAGG   TATCCTCT   AAGAGGCA   CTCTTATC   GGCAAAGA   CTCTTATC   CAAAGAGG   CTCTTATC   AAGAGGCA   TCCTCTTA   GGCAAAGA   TCCTCTTA   CAAAGAGG   TCCTCTTA      D11   AAGAGGCA   AGAGTAGA   GGCAAAGA   AGAGTAGA   CAAAGAGG   AGAGTAGA   AAGAGGCA   TAGAAGAG   GGCAAAGA   TAGAAGAG   CAAAGAGG   TAGAAGAG   AAGAGGCA   AGTAGAAG   GGCAAAGA   AGTAGAAG   CAAAGAGG   AGTAGAAG      E11   AAGAGGCA   GTAAGGAG   GGCAAAGA   GTAAGGAG   CAAAGAGG   GTAAGGAG   AAGAGGCA   GGAGGTAA   GGCAAAGA   GGAGGTAA   CAAAGAGG   GGAGGTAA   AAGAGGCA   AAGGAGGT   GGCAAAGA   AAGGAGGT   CAAAGAGG   AAGGAGGT      F11   AAGAGGCA   ACTGCATA   GGCAAAGA   ACTGCATA   CAAAGAGG   ACTGCATA   AAGAGGCA   CATAACTG   GGCAAAGA   CATAACTG   CAAAGAGG   CATAACTG   AAGAGGCA   TGCATAAC   GGCAAAGA   TGCATAAC   CAAAGAGG   TGCATAAC      G11   AAGAGGCA   AAGGAGTA   GGCAAAGA   AAGGAGTA   CAAAGAGG   AAGGAGTA   AAGAGGCA   AGTAAAGG   GGCAAAGA   AGTAAAGG   CAAAGAGG   AGTAAAGG   AAGAGGCA   GGAGTAAA   GGCAAAGA   GGAGTAAA   CAAAGAGG   GGAGTAAA      H11   AAGAGGCA   CTAAGCCT   GGCAAAGA   CTAAGCCT   CAAAGAGG   CTAAGCCT   AAGAGGCA   GCCTCTAA   GGCAAAGA   GCCTCTAA   CAAAGAGG   GCCTCTAA   AAGAGGCA   AAGCCTCT   GGCAAAGA   AAGCCTCT   CAAAGAGG   AAGCCTCT      A12   GTAGAGGA   TAGATCGC   AGGAGTAG   TAGATCGC   GAGTAGAG   TAGATCGC   GTAGAGGA   TCGCTAGA   AGGAGTAG   TCGCTAGA   GAGTAGAG   TCGCTAGA   GTAGAGGA   GATCGCTA   AGGAGTAG   GATCGCTA   GAGTAGAG   GATCGCTA      B12   GTAGAGGA   CTCTCTAT   AGGAGTAG   CTCTCTAT   GAGTAGAG   CTCTCTAT   GTAGAGGA   CTATCTCT   AGGAGTAG   CTATCTCT   GAGTAGAG   CTATCTCT   GTAGAGGA   CTCTATCT   AGGAGTAG   CTCTATCT   GAGTAGAG   CTCTATCT      C12   GTAGAGGA   TATCCTCT   AGGAGTAG   TATCCTCT   GAGTAGAG   TATCCTCT   GTAGAGGA   CTCTTATC   AGGAGTAG   CTCTTATC   GAGTAGAG   CTCTTATC   GTAGAGGA   TCCTCTTA   AGGAGTAG   TCCTCTTA   GAGTAGAG   TCCTCTTA      D12   GTAGAGGA   AGAGTAGA   AGGAGTAG   AGAGTAGA   GAGTAGAG   AGAGTAGA   GTAGAGGA   TAGAAGAG   AGGAGTAG   TAGAAGAG   GAGTAGAG   TAGAAGAG   GTAGAGGA   AGTAGAAG   AGGAGTAG   AGTAGAAG   GAGTAGAG   AGTAGAAG      E12   GTAGAGGA   GTAAGGAG   AGGAGTAG   GTAAGGAG   GAGTAGAG   GTAAGGAG   GTAGAGGA   GGAGGTAA   AGGAGTAG   GGAGGTAA   GAGTAGAG   GGAGGTAA   GTAGAGGA   AAGGAGGT   AGGAGTAG   AAGGAGGT   GAGTAGAG   AAGGAGGT      F12   GTAGAGGA   ACTGCATA   AGGAGTAG   ACTGCATA   GAGTAGAG   ACTGCATA   GTAGAGGA   CATAACTG   AGGAGTAG   CATAACTG   GAGTAGAG   CATAACTG   GTAGAGGA   TGCATAAC   AGGAGTAG   TGCATAAC   GAGTAGAG   TGCATAAC      G12   GTAGAGGA   AAGGAGTA   AGGAGTAG   AAGGAGTA   GAGTAGAG   AAGGAGTA   GTAGAGGA   AGTAAAGG   AGGAGTAG   AGTAAAGG   GAGTAGAG   AGTAAAGG   GTAGAGGA   GGAGTAAA   AGGAGTAG   GGAGTAAA   GAGTAGAG   GGAGTAAA      H12   GTAGAGGA   CTAAGCCT   AGGAGTAG   CTAAGCCT   GAGTAGAG   CTAAGCCT   GTAGAGGA   GCCTCTAA   AGGAGTAG   GCCTCTAA   GAGTAGAG   GCCTCTAA   GTAGAGGA   AAGCCTCT   AGGAGTAG   AAGCCTCT   GAGTAGAG   AAGCCTCT     
 
SECTION: Quality and quantity control of libraries
11. Determine the quantity of DNA available for each library
Determine the DNA concentration of each library following the instruction of the KAPA Library Quantification Kit Illumina® Platforms (Roche Sequencing Solutions).
Based on concentrations measured for each library, make a final pool of equimolar concentrations.

This final pool is ready for sequencing following Illumina instructions.