IDEEL- UNC Implementation of Targeted csp Sequencing- MIP to Nanopore

Jonathan Juliano; Jacob Sadler

Apr 13, 2026

Version 2

IDEEL- UNC Implementation of Targeted csp Sequencing- MIP to Nanopore V.2

DOI

https://dx.doi.org/10.17504/protocols.io.8epv55xz5v1b/v2

Jonathan Juliano¹,
Jacob Sadler²

¹Infectious Disease Epidemiology and Ecology Lab, University of North Carolina;
²Infectious Disease, Epidemiology, and Ecology Lab, University of North Carolina

IDEEL

Infectious Disease Epidemiology and Ecology Lab

University of North Carolina

DOI: https://dx.doi.org/10.17504/protocols.io.8epv55xz5v1b/v2

Protocol Citation: Jonathan Juliano, Jacob Sadler 2026. IDEEL- UNC Implementation of Targeted csp Sequencing- MIP to Nanopore. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55xz5v1b/v2Version created by Jacob Sadler

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: January 27, 2026

Last Modified: April 13, 2026

Protocol Integer ID: 241649

Keywords: PCR2 primer preparation, Pooling and Cleanup, Nanopore library preparation, Adaptor ligation, Nanopore sequencing, nanopore this protocol detail, amplicon generation through pcr2 primer preparation, nanopore library preparation, pcr2 primer preparation, nanopore, sequencing, amplicon generation, derived amplicon generation, unc implementation, mip, csp

Abstract

This protocol details the IDEEL-UNC targeted csp sequencing workflow, from MIP-derived amplicon generation through PCR2 primer preparation, pooling, cleanup, Nanopore library preparation, adaptor ligation, and sequencing.

Guidelines

Location of amplicon relative to csp:

Alignment of reverse primer

Materials

●    Molecular Grade Water (MGW)
●    Primers
○      sp-rtss-repeat_v4_F_MIP              GACTCGCCAAGCTGAAGNNNNTCGCAAACGTAATTAAATATTCACAAA
○      csp-rtss-repeat_v4_R_MIP            ACGTGTGCTCTTCCGATCTNNNNCCTTATTCCAGGAATACCAGTGC
●    GoTaq® Long PCR Master Mix, 2XPromegaCatalog #M402A  
●    Illumina FWD and REV barcode primers 
●    Gel electrophoresis reagents and equipment.
●    1X TE Low EDTA Buffer 
●    PromethION 2 solo and device specific flow cells
●    Ligation Sequencing Kit V14Oxford Nanopore TechnologiesCatalog #SQK-LSK114  

PCR2 Forward Primers

ABC
Well PositionNameSequence
A01hybrid_dual_fw_001AATGATACGGCGACCACCGAGATCTACACGTTAAGACACACTCTTTCCCTACACGACTCGCCAAGCTGA
A02hybrid_dual_fw_002AATGATACGGCGACCACCGAGATCTACACTCTAAGTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A03hybrid_dual_fw_003AATGATACGGCGACCACCGAGATCTACACTGTACATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A04hybrid_dual_fw_004AATGATACGGCGACCACCGAGATCTACACAATACCGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
A05hybrid_dual_fw_005AATGATACGGCGACCACCGAGATCTACACTGTCTATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
A06hybrid_dual_fw_006AATGATACGGCGACCACCGAGATCTACACTCACTGTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A07hybrid_dual_fw_007AATGATACGGCGACCACCGAGATCTACACTTGTATTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
A08hybrid_dual_fw_008AATGATACGGCGACCACCGAGATCTACACAGACTAACACACTCTTTCCCTACACGACTCGCCAAGCTGA
A09hybrid_dual_fw_009AATGATACGGCGACCACCGAGATCTACACATTGTCAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
A10hybrid_dual_fw_010AATGATACGGCGACCACCGAGATCTACACGTTCTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
A11hybrid_dual_fw_011AATGATACGGCGACCACCGAGATCTACACTTCATTCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
A12hybrid_dual_fw_012AATGATACGGCGACCACCGAGATCTACACAAGCGTCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B01hybrid_dual_fw_013AATGATACGGCGACCACCGAGATCTACACCGTAGGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
B02hybrid_dual_fw_014AATGATACGGCGACCACCGAGATCTACACAGTTAGCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
B03hybrid_dual_fw_015AATGATACGGCGACCACCGAGATCTACACGAACGTCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
B04hybrid_dual_fw_016AATGATACGGCGACCACCGAGATCTACACAGAACACTACACTCTTTCCCTACACGACTCGCCAAGCTGA
B05hybrid_dual_fw_017AATGATACGGCGACCACCGAGATCTACACTGTTGGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B06hybrid_dual_fw_018AATGATACGGCGACCACCGAGATCTACACGGCTAATAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B07hybrid_dual_fw_019AATGATACGGCGACCACCGAGATCTACACGCTAGAATACACTCTTTCCCTACACGACTCGCCAAGCTGA
B08hybrid_dual_fw_020AATGATACGGCGACCACCGAGATCTACACTGTACTAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B09hybrid_dual_fw_021AATGATACGGCGACCACCGAGATCTACACAGTTACACACACTCTTTCCCTACACGACTCGCCAAGCTGA
B10hybrid_dual_fw_022AATGATACGGCGACCACCGAGATCTACACGTACACGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B11hybrid_dual_fw_023AATGATACGGCGACCACCGAGATCTACACACTGTAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
B12hybrid_dual_fw_024AATGATACGGCGACCACCGAGATCTACACGATCATGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
C01hybrid_dual_fw_025AATGATACGGCGACCACCGAGATCTACACTGCTAACGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C02hybrid_dual_fw_026AATGATACGGCGACCACCGAGATCTACACTAATGCTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C03hybrid_dual_fw_027AATGATACGGCGACCACCGAGATCTACACCTTGTTGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
C04hybrid_dual_fw_028AATGATACGGCGACCACCGAGATCTACACGATACAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C05hybrid_dual_fw_029AATGATACGGCGACCACCGAGATCTACACATACAGCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C06hybrid_dual_fw_030AATGATACGGCGACCACCGAGATCTACACCTGGATAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C07hybrid_dual_fw_031AATGATACGGCGACCACCGAGATCTACACTCATCAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C08hybrid_dual_fw_032AATGATACGGCGACCACCGAGATCTACACTTATACCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C09hybrid_dual_fw_033AATGATACGGCGACCACCGAGATCTACACGCTGATTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C10hybrid_dual_fw_034AATGATACGGCGACCACCGAGATCTACACATAAGGCCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C11hybrid_dual_fw_035AATGATACGGCGACCACCGAGATCTACACAAGACTAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C12hybrid_dual_fw_036AATGATACGGCGACCACCGAGATCTACACTAGCAACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D01hybrid_dual_fw_037AATGATACGGCGACCACCGAGATCTACACCAAGTAATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D02hybrid_dual_fw_038AATGATACGGCGACCACCGAGATCTACACTCATGATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D03hybrid_dual_fw_039AATGATACGGCGACCACCGAGATCTACACTAGCTCTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
D04hybrid_dual_fw_040AATGATACGGCGACCACCGAGATCTACACACTACATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D05hybrid_dual_fw_041AATGATACGGCGACCACCGAGATCTACACTGTGCCATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D06hybrid_dual_fw_042AATGATACGGCGACCACCGAGATCTACACGACTTCCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D07hybrid_dual_fw_043AATGATACGGCGACCACCGAGATCTACACTATTCTCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D08hybrid_dual_fw_044AATGATACGGCGACCACCGAGATCTACACCACTAGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D09hybrid_dual_fw_045AATGATACGGCGACCACCGAGATCTACACCTTCGCATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D10hybrid_dual_fw_046AATGATACGGCGACCACCGAGATCTACACCTAGCGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D11hybrid_dual_fw_047AATGATACGGCGACCACCGAGATCTACACTACGTTAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D12hybrid_dual_fw_048AATGATACGGCGACCACCGAGATCTACACAAGTAGTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E01hybrid_dual_fw_049AATGATACGGCGACCACCGAGATCTACACGACGTATCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E02hybrid_dual_fw_050AATGATACGGCGACCACCGAGATCTACACACGTTCAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
E03hybrid_dual_fw_051AATGATACGGCGACCACCGAGATCTACACAGCTATATACACTCTTTCCCTACACGACTCGCCAAGCTGA
E04hybrid_dual_fw_052AATGATACGGCGACCACCGAGATCTACACAGTGCGAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E05hybrid_dual_fw_053AATGATACGGCGACCACCGAGATCTACACCAATGGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E06hybrid_dual_fw_054AATGATACGGCGACCACCGAGATCTACACATTCCGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
E07hybrid_dual_fw_055AATGATACGGCGACCACCGAGATCTACACAGCTGTTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
E08hybrid_dual_fw_056AATGATACGGCGACCACCGAGATCTACACTTCGACTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E09hybrid_dual_fw_057AATGATACGGCGACCACCGAGATCTACACCGAATGGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
E10hybrid_dual_fw_058AATGATACGGCGACCACCGAGATCTACACTAGTGTACACACTCTTTCCCTACACGACTCGCCAAGCTGA
E11hybrid_dual_fw_059AATGATACGGCGACCACCGAGATCTACACACGCATGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E12hybrid_dual_fw_060AATGATACGGCGACCACCGAGATCTACACTTCGTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F01hybrid_dual_fw_061AATGATACGGCGACCACCGAGATCTACACTTCTGCTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F02hybrid_dual_fw_062AATGATACGGCGACCACCGAGATCTACACTGGAAGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F03hybrid_dual_fw_063AATGATACGGCGACCACCGAGATCTACACGATGCTCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F04hybrid_dual_fw_064AATGATACGGCGACCACCGAGATCTACACAGCCTGAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F05hybrid_dual_fw_065AATGATACGGCGACCACCGAGATCTACACCACCAGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F06hybrid_dual_fw_066AATGATACGGCGACCACCGAGATCTACACGTTACACCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F07hybrid_dual_fw_067AATGATACGGCGACCACCGAGATCTACACGCGTTAGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
F08hybrid_dual_fw_068AATGATACGGCGACCACCGAGATCTACACTGACGTTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F09hybrid_dual_fw_069AATGATACGGCGACCACCGAGATCTACACTAGGCGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F10hybrid_dual_fw_070AATGATACGGCGACCACCGAGATCTACACAAGTGAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F11hybrid_dual_fw_071AATGATACGGCGACCACCGAGATCTACACCCGGAATAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F12hybrid_dual_fw_072AATGATACGGCGACCACCGAGATCTACACAATAGGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G01hybrid_dual_fw_073AATGATACGGCGACCACCGAGATCTACACTTCAGGTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G02hybrid_dual_fw_074AATGATACGGCGACCACCGAGATCTACACTAATCGCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
G03hybrid_dual_fw_075AATGATACGGCGACCACCGAGATCTACACCTTAGGTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G04hybrid_dual_fw_076AATGATACGGCGACCACCGAGATCTACACTTGATCTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G05hybrid_dual_fw_077AATGATACGGCGACCACCGAGATCTACACTCTTACTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G06hybrid_dual_fw_078AATGATACGGCGACCACCGAGATCTACACGAGTACACACACTCTTTCCCTACACGACTCGCCAAGCTGA
G07hybrid_dual_fw_079AATGATACGGCGACCACCGAGATCTACACACGTTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G08hybrid_dual_fw_080AATGATACGGCGACCACCGAGATCTACACTGGTAAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G09hybrid_dual_fw_081AATGATACGGCGACCACCGAGATCTACACGATAGCACACACTCTTTCCCTACACGACTCGCCAAGCTGA
G10hybrid_dual_fw_082AATGATACGGCGACCACCGAGATCTACACGACTTAGGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G11hybrid_dual_fw_083AATGATACGGCGACCACCGAGATCTACACAGTCGTTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G12hybrid_dual_fw_084AATGATACGGCGACCACCGAGATCTACACCGTGATTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
H01hybrid_dual_fw_085AATGATACGGCGACCACCGAGATCTACACTCAGTAGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
H02hybrid_dual_fw_086AATGATACGGCGACCACCGAGATCTACACATGTGTCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H03hybrid_dual_fw_087AATGATACGGCGACCACCGAGATCTACACCAGAATATACACTCTTTCCCTACACGACTCGCCAAGCTGA
H04hybrid_dual_fw_088AATGATACGGCGACCACCGAGATCTACACTAAGTCTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H05hybrid_dual_fw_089AATGATACGGCGACCACCGAGATCTACACATCGACATACACTCTTTCCCTACACGACTCGCCAAGCTGA
H06hybrid_dual_fw_090AATGATACGGCGACCACCGAGATCTACACACAGCTGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H07hybrid_dual_fw_091AATGATACGGCGACCACCGAGATCTACACATCACAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H08hybrid_dual_fw_092AATGATACGGCGACCACCGAGATCTACACGTCGAATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H09hybrid_dual_fw_093AATGATACGGCGACCACCGAGATCTACACTGCCGATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H10hybrid_dual_fw_094AATGATACGGCGACCACCGAGATCTACACACTAATGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
H11hybrid_dual_fw_095AATGATACGGCGACCACCGAGATCTACACCTAGTAGGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H12hybrid_dual_fw_096AATGATACGGCGACCACCGAGATCTACACTTAAGCCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
 
PCR2 Reverse Primers
 
Well PositionNameSequence
A01hybrid_dual_rev_001CAAGCAGAAGACGGCATACGAGATGTTAAGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A02hybrid_dual_rev_002CAAGCAGAAGACGGCATACGAGATTCTAAGTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A03hybrid_dual_rev_003CAAGCAGAAGACGGCATACGAGATTGTACATTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A04hybrid_dual_rev_004CAAGCAGAAGACGGCATACGAGATAATACCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A05hybrid_dual_rev_005CAAGCAGAAGACGGCATACGAGATTGTCTATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A06hybrid_dual_rev_006CAAGCAGAAGACGGCATACGAGATTCACTGTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A07hybrid_dual_rev_007CAAGCAGAAGACGGCATACGAGATTTGTATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A08hybrid_dual_rev_008CAAGCAGAAGACGGCATACGAGATAGACTAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A09hybrid_dual_rev_009CAAGCAGAAGACGGCATACGAGATATTGTCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A10hybrid_dual_rev_010CAAGCAGAAGACGGCATACGAGATGTTCTACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A11hybrid_dual_rev_011CAAGCAGAAGACGGCATACGAGATTTCATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A12hybrid_dual_rev_012CAAGCAGAAGACGGCATACGAGATAAGCGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B01hybrid_dual_rev_013CAAGCAGAAGACGGCATACGAGATCGTAGGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B02hybrid_dual_rev_014CAAGCAGAAGACGGCATACGAGATAGTTAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B03hybrid_dual_rev_015CAAGCAGAAGACGGCATACGAGATGAACGTCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B04hybrid_dual_rev_016CAAGCAGAAGACGGCATACGAGATAGAACACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B05hybrid_dual_rev_017CAAGCAGAAGACGGCATACGAGATTGTTGGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B06hybrid_dual_rev_018CAAGCAGAAGACGGCATACGAGATGGCTAATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B07hybrid_dual_rev_019CAAGCAGAAGACGGCATACGAGATGCTAGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B08hybrid_dual_rev_020CAAGCAGAAGACGGCATACGAGATTGTACTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B09hybrid_dual_rev_021CAAGCAGAAGACGGCATACGAGATAGTTACACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B10hybrid_dual_rev_022CAAGCAGAAGACGGCATACGAGATGTACACGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B11hybrid_dual_rev_023CAAGCAGAAGACGGCATACGAGATACTGTAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B12hybrid_dual_rev_024CAAGCAGAAGACGGCATACGAGATGATCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C01hybrid_dual_rev_025CAAGCAGAAGACGGCATACGAGATTGCTAACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C02hybrid_dual_rev_026CAAGCAGAAGACGGCATACGAGATTAATGCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C03hybrid_dual_rev_027CAAGCAGAAGACGGCATACGAGATCTTGTTGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C04hybrid_dual_rev_028CAAGCAGAAGACGGCATACGAGATGATACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C05hybrid_dual_rev_029CAAGCAGAAGACGGCATACGAGATATACAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C06hybrid_dual_rev_030CAAGCAGAAGACGGCATACGAGATCTGGATAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C07hybrid_dual_rev_031CAAGCAGAAGACGGCATACGAGATTCATCAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C08hybrid_dual_rev_032CAAGCAGAAGACGGCATACGAGATTTATACCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C09hybrid_dual_rev_033CAAGCAGAAGACGGCATACGAGATGCTGATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C10hybrid_dual_rev_034CAAGCAGAAGACGGCATACGAGATATAAGGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C11hybrid_dual_rev_035CAAGCAGAAGACGGCATACGAGATAAGACTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C12hybrid_dual_rev_036CAAGCAGAAGACGGCATACGAGATTAGCAACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D01hybrid_dual_rev_037CAAGCAGAAGACGGCATACGAGATCAAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D02hybrid_dual_rev_038CAAGCAGAAGACGGCATACGAGATTCATGATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D03hybrid_dual_rev_039CAAGCAGAAGACGGCATACGAGATTAGCTCTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D04hybrid_dual_rev_040CAAGCAGAAGACGGCATACGAGATACTACATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D05hybrid_dual_rev_041CAAGCAGAAGACGGCATACGAGATTGTGCCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D06hybrid_dual_rev_042CAAGCAGAAGACGGCATACGAGATGACTTCCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D07hybrid_dual_rev_043CAAGCAGAAGACGGCATACGAGATTATTCTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D08hybrid_dual_rev_044CAAGCAGAAGACGGCATACGAGATCACTAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D09hybrid_dual_rev_045CAAGCAGAAGACGGCATACGAGATCTTCGCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D10hybrid_dual_rev_046CAAGCAGAAGACGGCATACGAGATCTAGCGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D11hybrid_dual_rev_047CAAGCAGAAGACGGCATACGAGATTACGTTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D12hybrid_dual_rev_048CAAGCAGAAGACGGCATACGAGATAAGTAGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E01hybrid_dual_rev_049CAAGCAGAAGACGGCATACGAGATGACGTATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E02hybrid_dual_rev_050CAAGCAGAAGACGGCATACGAGATACGTTCAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E03hybrid_dual_rev_051CAAGCAGAAGACGGCATACGAGATAGCTATATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E04hybrid_dual_rev_052CAAGCAGAAGACGGCATACGAGATAGTGCGAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E05hybrid_dual_rev_053CAAGCAGAAGACGGCATACGAGATCAATGGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E06hybrid_dual_rev_054CAAGCAGAAGACGGCATACGAGATATTCCGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E07hybrid_dual_rev_055CAAGCAGAAGACGGCATACGAGATAGCTGTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E08hybrid_dual_rev_056CAAGCAGAAGACGGCATACGAGATTTCGACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E09hybrid_dual_rev_057CAAGCAGAAGACGGCATACGAGATCGAATGGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E10hybrid_dual_rev_058CAAGCAGAAGACGGCATACGAGATTAGTGTACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E11hybrid_dual_rev_059CAAGCAGAAGACGGCATACGAGATACGCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
E12hybrid_dual_rev_060CAAGCAGAAGACGGCATACGAGATTTCGTACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F01hybrid_dual_rev_061CAAGCAGAAGACGGCATACGAGATTTCTGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F02hybrid_dual_rev_062CAAGCAGAAGACGGCATACGAGATTGGAAGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F03hybrid_dual_rev_063CAAGCAGAAGACGGCATACGAGATGATGCTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F04hybrid_dual_rev_064CAAGCAGAAGACGGCATACGAGATAGCCTGAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F05hybrid_dual_rev_065CAAGCAGAAGACGGCATACGAGATCACCAGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F06hybrid_dual_rev_066CAAGCAGAAGACGGCATACGAGATGTTACACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F07hybrid_dual_rev_067CAAGCAGAAGACGGCATACGAGATGCGTTAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F08hybrid_dual_rev_068CAAGCAGAAGACGGCATACGAGATTGACGTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F09hybrid_dual_rev_069CAAGCAGAAGACGGCATACGAGATTAGGCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F10hybrid_dual_rev_070CAAGCAGAAGACGGCATACGAGATAAGTGAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F11hybrid_dual_rev_071CAAGCAGAAGACGGCATACGAGATCCGGAATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
F12hybrid_dual_rev_072CAAGCAGAAGACGGCATACGAGATAATAGGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G01hybrid_dual_rev_073CAAGCAGAAGACGGCATACGAGATTTCAGGTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G02hybrid_dual_rev_074CAAGCAGAAGACGGCATACGAGATTAATCGCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G03hybrid_dual_rev_075CAAGCAGAAGACGGCATACGAGATCTTAGGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G04hybrid_dual_rev_076CAAGCAGAAGACGGCATACGAGATTTGATCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G05hybrid_dual_rev_077CAAGCAGAAGACGGCATACGAGATTCTTACTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G06hybrid_dual_rev_078CAAGCAGAAGACGGCATACGAGATGAGTACACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G07hybrid_dual_rev_079CAAGCAGAAGACGGCATACGAGATACGTTACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G08hybrid_dual_rev_080CAAGCAGAAGACGGCATACGAGATTGGTAAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G09hybrid_dual_rev_081CAAGCAGAAGACGGCATACGAGATGATAGCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G10hybrid_dual_rev_082CAAGCAGAAGACGGCATACGAGATGACTTAGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G11hybrid_dual_rev_083CAAGCAGAAGACGGCATACGAGATAGTCGTTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
G12hybrid_dual_rev_084CAAGCAGAAGACGGCATACGAGATCGTGATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H01hybrid_dual_rev_085CAAGCAGAAGACGGCATACGAGATTCAGTAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H02hybrid_dual_rev_086CAAGCAGAAGACGGCATACGAGATATGTGTCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H03hybrid_dual_rev_087CAAGCAGAAGACGGCATACGAGATCAGAATATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H04hybrid_dual_rev_088CAAGCAGAAGACGGCATACGAGATTAAGTCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H05hybrid_dual_rev_089CAAGCAGAAGACGGCATACGAGATATCGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H06hybrid_dual_rev_090CAAGCAGAAGACGGCATACGAGATACAGCTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H07hybrid_dual_rev_091CAAGCAGAAGACGGCATACGAGATATCACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H08hybrid_dual_rev_092CAAGCAGAAGACGGCATACGAGATGTCGAATTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H09hybrid_dual_rev_093CAAGCAGAAGACGGCATACGAGATTGCCGATTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H10hybrid_dual_rev_094CAAGCAGAAGACGGCATACGAGATACTAATGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H11hybrid_dual_rev_095CAAGCAGAAGACGGCATACGAGATCTAGTAGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
H12hybrid_dual_rev_096CAAGCAGAAGACGGCATACGAGATTTAAGCCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
 

Preparation of PCR2 Primer plates

To allow for high levels of combinations of indexing primers, we generate multiple forward primer plates (12) from a single 96-well plate. 

By using these 12 plates in combination with a single 96-well plate of reverse primers, we can index 1,152 samples. 

Additional primers can be designed to increase this multiplexing. Here we present one plate of forward and one plate of reverse primers to allow for this level of combination.

To generate forward primer plates, we shift the column to the right one column when replating to a new plate (e.g. column 1 becomes column 2 and column 12 becomes column 1). See the figure below. Be sure to uniquely label the plate so you know which primers are used in library preparation.

Note
We do NOT do this for reverse primers.

Generate Primers mix

Spin lyophilized Primer.

Resuspend in MGW using the provided volume for a 100 micromolar (µM)   stock.

Vortex and spin down briefly.

Let the mixture sit for about 00:10:00   for complete dissolution.

Prepare the stock primer pool (100 micromolar (µM)  ) by mixing 1 volume of sp-rtss-repeat_v4_F_MIP with 1 volume of csp-rtss-repeat_v4_R_MIP.

Dilute primer pool with MGW to obtain 20 micromolar (µM)   working solution: 1 volume of stock + 4 volumes of MGW.

PCR1

Pre-set the thermocycler before starting master mix preparation using the programme below.

ABCD
  Step
  Temperature (oC)  Time (minute)
    Number of cycles
  
  Step1 (preheat)
    95
  HoldNA
  Step2 (Activation)
    95
  02:00NA
  Step3 (Denaturation)
    94
  00:30x32
  Step4 (Annealing)
  6506:00
  Step5 (Final Extension)
    72
    10
  NA
  Step6
    4
    hold
  NA
Step 3 and 4 are a dual anneal and extend step (no need for separate extension step until final extension)

 

All sample plates and controls are to be kept on cold blocks - switch out cold blocks if they are getting warm.

All reagents are to be kept On ice   and the tube used to prepare the master mix should also remain On ice  .

Centrifuge sample plates at 4000 rpm, 00:01:00 .

Prepare the master mix using the template (here 96 reactions) below and adjust it to the number of reactions.

### These volumes could be halved but not tested at that volume. ###

ABC
Master mix x1
  x96* 1.1
Gotaq252,640
Molecular grade water 192,606.4
Primer pool (sp-rtss-repeat_v4_F_MIP & csp-rtss-repeat_v4_R_MIP)1105.6
Template DNA5

Divide the total master mix volume by 12 and aliquot the designated volume of into 12 tube strip on cold block to allow for multichannel pipetting.

Using a multichannel, pipette 45 µL   of master mix into each well of 96 well plate..

Using a multichannel, plate 5 µL   of sample into each well on the PCR1 plate.

Add at least one positive control (lab strain, 5 µL  ) and one PCR1 negative control (MGW, 5 µL  ).

Centrifuge the plate at 4000 rpm, 00:01:00 .

Place the plate in the thermocycler and start running the pre-set PCR program.

PCR2 or barcoding PCR

Once the first PCR is complete, pre-set the thermocycler before starting the master mix preparation for PCR2 using the program below.
 
ABCD
  Step
  Temperature (oC)  Time (minute)
    Number of cycles
  
  Step1
    98
    hold
    NA
  
  Step2
    98
    0:30
    NA
  
  Step3
    95
    0:10
    x10
  
  Step4
    62
    6:0
  
  Step5
    72
    0:20
  
  Step6
    72
    1:0
    NA
  
  Step7
    4
    hold
    NA
  
 

Prepare the master mix for PCR2 using the template (here 96 reactions) below and adjust it to the number of reactions. 
 
ABC
  Master mix
    x1
  x96* 1.1
  Gotaq
    10
    1056
  
  Molecular grade water
    6
    633.6
  
 

Divide the total master mix volume by 12 and aliquot the designated volume (140.8 µL   for 96 sample run) into 12 tube strip on cold block to allow for multichannel pipetting.

Using a multichannel, pipette 16 µL   of master mix into each well.

Spin down the forward and reverse PCR2 primer plates needed for the experiment at 4000 rpm, 00:01:00  and add 1 µL   of forward primer and 1 µL   of reverse primer into each well using the multichannel. 

On your sample sheet, please denote the name of the forward and reverse primer plate used and the well of the sample to be sure you know the correct index for the sample you are genotyping.

In the post-PCR lab, add 2 µL   of the PCR1 product to the master mix using a multichannel.

Optional: shake the plate gently on a shaker for 00:01:00   to mix the master mix added to the samples.

Centrifuge the plate at 4000 rpm, 00:01:00 .

Place the plate in the thermocycler and start running the pre-set PCR program.

Check a random set of 10 wells per plate for amplification by gel electrophoresis based on your lab protocol (including the NTC and positive control).

PCR Product Pooling and Cleanup

10m

Pool 5 µL   of PCR2 product into a single Eppendorf tube. If the pool is very large, 3 µL   is sufficient.

Bring Ampure Beads to Room temperature  . This can also be done with SpeedBead Mix (DIY).

Add an equal volume of beads to the pool (1X bead clean). Let it incubate for at least 00:10:00   at Room temperature  . For a higher yield, place the tube on a Hula Mixer or invert by hand.

Place the pool on a magnet and wait until the beads have pelletted on the magnet and the solution is clear. Prepare enough fresh 80% ethanol to cover the beads for 2 washes.

Remove the supernatant and reserve it in another tube.

Wash by dispensing enough 80% ethanol to cover entire bead pellet. Incubate for 00:00:30  . Aspirate and discard the ethanol.

Repeat the previous step for a second wash.

Remove the ethanol, then aspirate final ethanol drops with a P20 pipette. Let the beads dry for around 00:01:00   (do not let the beads dry until cracking). 

If the pool was large, it could take over 1 minute to dry properly.

Elute in 51 µL   of 1X low EDTA TE Buffer. If the pool is very large, use your discretion and elute in a higher volume (we eluted in 150 µL   with 6 96-well plates of sample). Incubate at Room temperature   for 00:10:00  .

10m

Pellet the beads on a magnet and remove the elution into a clean tube.

Quantify the elution with a Qubit fluorometer.

Nanopore Library preparation

We use the SQK-LSK114 Kit (Ligation Sequencing) and have adapted this protocol from the Ligation Sequencing Amplicons V14 protocol from Nanopore. We use the P2 sequencing machine, but this protocol could be adapted for any Nanopore platform.

Nanopore Library preparation - End Prep

26m 30s

Transfer 200 fmol of cleaned DNA into a PCR tube or plate. Calculation for fmol can be done on this website: https://www.promega.com/resources/tools/biomath/ (1 pmol = 1,000 fmol). Adjust the volume to 50 µL   with nuclease-free water. 

This is approximately 240 nanograms in 50uL for the ~1.8kb fragment

Bring SPRI beads to Room temperature  

Add the following to a PCR tube:
 
AB
  Reagent
    Volume
  
  DNA Template
    50 µL
  Ultra II End Prep Reaction Buffer
    7 µL
  
  Ultra II End Prep Enzyme Mix
    3 µL
  
  Total
    60µL
  
 

Mix thoroughly by pipetting.

Incubate at 20 °C   for 00:05:00   and 65 °C   for 00:05:00  .

10m

Spin down and transfer the sample to a clean 1.5ml Eppendorf tube.

 Vortedt SPRI beads thoroughly then add 60 µL   of beads to the DNA sample. Mix by flicking tube and spin down briefly.

Incubate for 00:10:00   at Room temperature  . For increased yield, place the tube on a Hula Mixer or invert by hand. Meanwhile, prepare 1 mL   of 80% ethanol.

10m

Pellet on a magnet and remove the supernatant - keep the supernatant for extra security.

Wash with 500 µL   of 80% ethanol. Incubate at room temperature for 00:00:30  . Aspirate ethanol and discard.

30s

Repeat the previous step for a total of 2 washes.

Let the beads dry for around 00:01:00  . Do not let the beads crack on the side of the tube.

Elute in 61 µL   of nuclease-free water. Let it incubate at Room temperature   for 00:05:00  .

Pellet on the magnet and transfer the eluate to a clean tube.

Quantify the end-prepped DNA with a qubit fluorometer. This library can be stored at 4 °C   short-term, or at -20 °C   or -80 °C   for long-term storage.

Nanopore Library preparation - Adapter Ligation

21m 30s

Thaw the Short Fragment Buffer (SFB), Elution Buffer (EB) and SPRI beads to Room temperature  .  Room temperature  . Place the Ligation Adapter (LA), Ligation Buffer (LNB), and Quick T4 Ligase On ice  .

Mix the following in a 1.5 ml Eppendorf tube, mixing 10-20 times after each addition by pipette: 
 
AB
  Reagent
    Volume
  
  End-prepped DNA
    60 µL
  
  Ligation Buffer (LNB)
    25 µL
  
  NEBNext Quick T4 Ligase
    10 µL
  
  Ligation Adapter (LA)
    5 µL
  
  Total
    100 µL
  
 

Incubate at Room temperature   for at least 00:10:00  . A longer incubation of 00:20:00   will increase the amount of ligated adapters.

Vortex SPRI beads thoroughly.

Add 60 µL   of beads and mix by pipette. Incubate for 00:10:00   at Room temperature  . For higher yield, invert in your hand or place on a Hula mixer

10m

Pellet on a magnet and remove the supernatant.

Wash with 250 µL   of the Short Fragment buffer. Remove after around 00:00:30  .

30s

Repeat the previous step for a total of 2 washes.

Allow beads to dry on the magnet, and pipette out any residual buffer. Do not let beads dry to the point of cracking.

Resuspend in 25 µL   of the Elution Buffer (EB). Incubate for 00:10:00   at Room temperature  .

10m

Pellet on a magnet and pipette the eluate into clean tube.

Quantify the library with a qubit fluorometer. Be sure that you have at least 120 fmol of DNA, which is around 25 ng   for a 4CAST library. This library can be stored at 4 °C   for short-term storage, or at -20 °C   or -80 °C   for long-term storage.

Nanopore sequencing - Loading the Flow Cell

20m

Take the Promethion Flow Cell you plan to use out of the 4 °C   refrigerator at least 00:20:00   before use. It is highly recommended to do a flow cell check before any sequencing.

Determine 100 - 120 fmols of library and add to an Eppendorf tube (Nanopore recommends 20 fmol for sequencing; however, this leads to underloading of the flow cell in most cases. If you have less than 100 fmols, add the entire library). 25 ng   of CSP Library is around 120 fmols.

Bring up the 100 - 120 fmols of library to 32 µL   with the elution buffer (EB).

After 00:20:00   at Room temperature  , gently insert the flow cell into one of the ports of the P2. The lights will turn blue if inserted correctly, and red if inserted incorrectly.

In a 1.5 Eppendorf tube, mix the following to create the flow cell priming mix:
 
AB
  Reagent
    Volume
  
  Flow Cell Flush (FCF)
    1170 µL
  
  Flow Cell Tether (FCT)
    30 µL
  
  Total
    1200 µL
  
 

Set a P1000 pipette to 200. Open the inlet port and place the tip inside the port. Turn the dial until it shows 220-230. A small amount of buffer should enter the tip. Discard the tip and buffer.

Load 500 µL   of the flow cell priming mix into the inlet port. Load by slowly twisting the dial in the counterclockwise direction. Wait 00:05:00  .

While the priming mix is incubating, prepare the library:
 
AB
  Reagent
    Volume
  
  Sequencing Buffer (SB)
    100 µL
  
  Library Beads (LIB)
    68 µL
  
  DNA Library
    32 µL
  
  Total
    200 µL
  
 

After the 00:05:00   incubation, add another 500 µL   of flow cell priming mix to the inlet port.

Immediately add the 200 µL   of the library slowly into the inlet port.

Close the inlet port and wait for the library to incubate for 00:10:00   before starting any sequencing.

10m

Nanopore sequencing - Flow cell wash

A flow cell wash should be done immediately after the sequencing run is complete.

Place the Wash Mix (WMX) On ice  , and thaw the Wash Diluent (DIL) at Room temperature  .

Vortex the Wash Diluent thoroughly.

Make the Flow Cell Wash Mix by adding the following to a 1.5ml Eppendorf tube:  
 
AB
  Reagent
    Volume
  
  Wash Diluent (DIL)
    398 µL
  
  Wash Mix (WMX)
    2 µL
  
 

Pipette to mix and place On ice  .

Stop the sequencing run on Minknow, and leave the flow cell in the port.

Make sure the inlet port is closed, and remove waste from port 2 or port 3 with a P1000 pipette.

Open the inlet port and set the P1000 to 200. Remove the air bubble by turning the wheel until it reaches 220-230, and a small volume of buffer enters the pipette tip.

Load 400 µL   of the Flow Cell Wash Mix into the inlet port. Incubate for 01:00:00  .

After 01:00:00  , remove the waste again from ports 2 or 3. Make sure the inlet port is closed.

Remove the air bubble from the inlet port if it is present after removing the waste. Then add 500 µL   of Storage Buffer (S). The flow cell can now be placed back into a 4 °C   refrigerator for long-term storage. Incubate the flow cell at least Overnight   before doing another flow cell check. 

A	B	C
Well Position	Name	Sequence
A01	hybrid_dual_fw_001	AATGATACGGCGACCACCGAGATCTACACGTTAAGACACACTCTTTCCCTACACGACTCGCCAAGCTGA
A02	hybrid_dual_fw_002	AATGATACGGCGACCACCGAGATCTACACTCTAAGTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A03	hybrid_dual_fw_003	AATGATACGGCGACCACCGAGATCTACACTGTACATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A04	hybrid_dual_fw_004	AATGATACGGCGACCACCGAGATCTACACAATACCGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
A05	hybrid_dual_fw_005	AATGATACGGCGACCACCGAGATCTACACTGTCTATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
A06	hybrid_dual_fw_006	AATGATACGGCGACCACCGAGATCTACACTCACTGTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
A07	hybrid_dual_fw_007	AATGATACGGCGACCACCGAGATCTACACTTGTATTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
A08	hybrid_dual_fw_008	AATGATACGGCGACCACCGAGATCTACACAGACTAACACACTCTTTCCCTACACGACTCGCCAAGCTGA
A09	hybrid_dual_fw_009	AATGATACGGCGACCACCGAGATCTACACATTGTCAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
A10	hybrid_dual_fw_010	AATGATACGGCGACCACCGAGATCTACACGTTCTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
A11	hybrid_dual_fw_011	AATGATACGGCGACCACCGAGATCTACACTTCATTCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
A12	hybrid_dual_fw_012	AATGATACGGCGACCACCGAGATCTACACAAGCGTCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B01	hybrid_dual_fw_013	AATGATACGGCGACCACCGAGATCTACACCGTAGGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
B02	hybrid_dual_fw_014	AATGATACGGCGACCACCGAGATCTACACAGTTAGCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
B03	hybrid_dual_fw_015	AATGATACGGCGACCACCGAGATCTACACGAACGTCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
B04	hybrid_dual_fw_016	AATGATACGGCGACCACCGAGATCTACACAGAACACTACACTCTTTCCCTACACGACTCGCCAAGCTGA
B05	hybrid_dual_fw_017	AATGATACGGCGACCACCGAGATCTACACTGTTGGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B06	hybrid_dual_fw_018	AATGATACGGCGACCACCGAGATCTACACGGCTAATAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B07	hybrid_dual_fw_019	AATGATACGGCGACCACCGAGATCTACACGCTAGAATACACTCTTTCCCTACACGACTCGCCAAGCTGA
B08	hybrid_dual_fw_020	AATGATACGGCGACCACCGAGATCTACACTGTACTAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B09	hybrid_dual_fw_021	AATGATACGGCGACCACCGAGATCTACACAGTTACACACACTCTTTCCCTACACGACTCGCCAAGCTGA
B10	hybrid_dual_fw_022	AATGATACGGCGACCACCGAGATCTACACGTACACGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
B11	hybrid_dual_fw_023	AATGATACGGCGACCACCGAGATCTACACACTGTAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
B12	hybrid_dual_fw_024	AATGATACGGCGACCACCGAGATCTACACGATCATGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
C01	hybrid_dual_fw_025	AATGATACGGCGACCACCGAGATCTACACTGCTAACGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C02	hybrid_dual_fw_026	AATGATACGGCGACCACCGAGATCTACACTAATGCTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C03	hybrid_dual_fw_027	AATGATACGGCGACCACCGAGATCTACACCTTGTTGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
C04	hybrid_dual_fw_028	AATGATACGGCGACCACCGAGATCTACACGATACAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C05	hybrid_dual_fw_029	AATGATACGGCGACCACCGAGATCTACACATACAGCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C06	hybrid_dual_fw_030	AATGATACGGCGACCACCGAGATCTACACCTGGATAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C07	hybrid_dual_fw_031	AATGATACGGCGACCACCGAGATCTACACTCATCAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C08	hybrid_dual_fw_032	AATGATACGGCGACCACCGAGATCTACACTTATACCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C09	hybrid_dual_fw_033	AATGATACGGCGACCACCGAGATCTACACGCTGATTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C10	hybrid_dual_fw_034	AATGATACGGCGACCACCGAGATCTACACATAAGGCCACACTCTTTCCCTACACGACTCGCCAAGCTGA
C11	hybrid_dual_fw_035	AATGATACGGCGACCACCGAGATCTACACAAGACTAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
C12	hybrid_dual_fw_036	AATGATACGGCGACCACCGAGATCTACACTAGCAACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D01	hybrid_dual_fw_037	AATGATACGGCGACCACCGAGATCTACACCAAGTAATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D02	hybrid_dual_fw_038	AATGATACGGCGACCACCGAGATCTACACTCATGATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D03	hybrid_dual_fw_039	AATGATACGGCGACCACCGAGATCTACACTAGCTCTTACACTCTTTCCCTACACGACTCGCCAAGCTGA
D04	hybrid_dual_fw_040	AATGATACGGCGACCACCGAGATCTACACACTACATGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D05	hybrid_dual_fw_041	AATGATACGGCGACCACCGAGATCTACACTGTGCCATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D06	hybrid_dual_fw_042	AATGATACGGCGACCACCGAGATCTACACGACTTCCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D07	hybrid_dual_fw_043	AATGATACGGCGACCACCGAGATCTACACTATTCTCGACACTCTTTCCCTACACGACTCGCCAAGCTGA
D08	hybrid_dual_fw_044	AATGATACGGCGACCACCGAGATCTACACCACTAGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D09	hybrid_dual_fw_045	AATGATACGGCGACCACCGAGATCTACACCTTCGCATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D10	hybrid_dual_fw_046	AATGATACGGCGACCACCGAGATCTACACCTAGCGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
D11	hybrid_dual_fw_047	AATGATACGGCGACCACCGAGATCTACACTACGTTAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
D12	hybrid_dual_fw_048	AATGATACGGCGACCACCGAGATCTACACAAGTAGTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E01	hybrid_dual_fw_049	AATGATACGGCGACCACCGAGATCTACACGACGTATCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E02	hybrid_dual_fw_050	AATGATACGGCGACCACCGAGATCTACACACGTTCAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
E03	hybrid_dual_fw_051	AATGATACGGCGACCACCGAGATCTACACAGCTATATACACTCTTTCCCTACACGACTCGCCAAGCTGA
E04	hybrid_dual_fw_052	AATGATACGGCGACCACCGAGATCTACACAGTGCGAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E05	hybrid_dual_fw_053	AATGATACGGCGACCACCGAGATCTACACCAATGGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E06	hybrid_dual_fw_054	AATGATACGGCGACCACCGAGATCTACACATTCCGATACACTCTTTCCCTACACGACTCGCCAAGCTGA
E07	hybrid_dual_fw_055	AATGATACGGCGACCACCGAGATCTACACAGCTGTTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
E08	hybrid_dual_fw_056	AATGATACGGCGACCACCGAGATCTACACTTCGACTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
E09	hybrid_dual_fw_057	AATGATACGGCGACCACCGAGATCTACACCGAATGGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
E10	hybrid_dual_fw_058	AATGATACGGCGACCACCGAGATCTACACTAGTGTACACACTCTTTCCCTACACGACTCGCCAAGCTGA
E11	hybrid_dual_fw_059	AATGATACGGCGACCACCGAGATCTACACACGCATGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
E12	hybrid_dual_fw_060	AATGATACGGCGACCACCGAGATCTACACTTCGTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F01	hybrid_dual_fw_061	AATGATACGGCGACCACCGAGATCTACACTTCTGCTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F02	hybrid_dual_fw_062	AATGATACGGCGACCACCGAGATCTACACTGGAAGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F03	hybrid_dual_fw_063	AATGATACGGCGACCACCGAGATCTACACGATGCTCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F04	hybrid_dual_fw_064	AATGATACGGCGACCACCGAGATCTACACAGCCTGAAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F05	hybrid_dual_fw_065	AATGATACGGCGACCACCGAGATCTACACCACCAGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F06	hybrid_dual_fw_066	AATGATACGGCGACCACCGAGATCTACACGTTACACCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F07	hybrid_dual_fw_067	AATGATACGGCGACCACCGAGATCTACACGCGTTAGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
F08	hybrid_dual_fw_068	AATGATACGGCGACCACCGAGATCTACACTGACGTTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F09	hybrid_dual_fw_069	AATGATACGGCGACCACCGAGATCTACACTAGGCGTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F10	hybrid_dual_fw_070	AATGATACGGCGACCACCGAGATCTACACAAGTGAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
F11	hybrid_dual_fw_071	AATGATACGGCGACCACCGAGATCTACACCCGGAATAACACTCTTTCCCTACACGACTCGCCAAGCTGA
F12	hybrid_dual_fw_072	AATGATACGGCGACCACCGAGATCTACACAATAGGCAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G01	hybrid_dual_fw_073	AATGATACGGCGACCACCGAGATCTACACTTCAGGTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G02	hybrid_dual_fw_074	AATGATACGGCGACCACCGAGATCTACACTAATCGCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
G03	hybrid_dual_fw_075	AATGATACGGCGACCACCGAGATCTACACCTTAGGTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G04	hybrid_dual_fw_076	AATGATACGGCGACCACCGAGATCTACACTTGATCTCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G05	hybrid_dual_fw_077	AATGATACGGCGACCACCGAGATCTACACTCTTACTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G06	hybrid_dual_fw_078	AATGATACGGCGACCACCGAGATCTACACGAGTACACACACTCTTTCCCTACACGACTCGCCAAGCTGA
G07	hybrid_dual_fw_079	AATGATACGGCGACCACCGAGATCTACACACGTTACAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G08	hybrid_dual_fw_080	AATGATACGGCGACCACCGAGATCTACACTGGTAAGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
G09	hybrid_dual_fw_081	AATGATACGGCGACCACCGAGATCTACACGATAGCACACACTCTTTCCCTACACGACTCGCCAAGCTGA
G10	hybrid_dual_fw_082	AATGATACGGCGACCACCGAGATCTACACGACTTAGGACACTCTTTCCCTACACGACTCGCCAAGCTGA
G11	hybrid_dual_fw_083	AATGATACGGCGACCACCGAGATCTACACAGTCGTTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
G12	hybrid_dual_fw_084	AATGATACGGCGACCACCGAGATCTACACCGTGATTAACACTCTTTCCCTACACGACTCGCCAAGCTGA
H01	hybrid_dual_fw_085	AATGATACGGCGACCACCGAGATCTACACTCAGTAGAACACTCTTTCCCTACACGACTCGCCAAGCTGA
H02	hybrid_dual_fw_086	AATGATACGGCGACCACCGAGATCTACACATGTGTCTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H03	hybrid_dual_fw_087	AATGATACGGCGACCACCGAGATCTACACCAGAATATACACTCTTTCCCTACACGACTCGCCAAGCTGA
H04	hybrid_dual_fw_088	AATGATACGGCGACCACCGAGATCTACACTAAGTCTGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H05	hybrid_dual_fw_089	AATGATACGGCGACCACCGAGATCTACACATCGACATACACTCTTTCCCTACACGACTCGCCAAGCTGA
H06	hybrid_dual_fw_090	AATGATACGGCGACCACCGAGATCTACACACAGCTGTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H07	hybrid_dual_fw_091	AATGATACGGCGACCACCGAGATCTACACATCACAAGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H08	hybrid_dual_fw_092	AATGATACGGCGACCACCGAGATCTACACGTCGAATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H09	hybrid_dual_fw_093	AATGATACGGCGACCACCGAGATCTACACTGCCGATTACACTCTTTCCCTACACGACTCGCCAAGCTGA
H10	hybrid_dual_fw_094	AATGATACGGCGACCACCGAGATCTACACACTAATGCACACTCTTTCCCTACACGACTCGCCAAGCTGA
H11	hybrid_dual_fw_095	AATGATACGGCGACCACCGAGATCTACACCTAGTAGGACACTCTTTCCCTACACGACTCGCCAAGCTGA
H12	hybrid_dual_fw_096	AATGATACGGCGACCACCGAGATCTACACTTAAGCCAACACTCTTTCCCTACACGACTCGCCAAGCTGA

A	B	C	D
Step	Temperature (^oC)	Time (minute)	Number of cycles
Step1 (preheat)	95	Hold	NA
Step2 (Activation)	95	02:00	NA
Step3 (Denaturation)	94	00:30	x32
Step4 (Annealing)	65	06:00	x32
Step5 (Final Extension)	72	10	NA
Step6	4	hold	NA

A	B	C
Master mix	x1	x96^*1.1
Gotaq	25	2,640
Molecular grade water	19	2,606.4
Primer pool (sp-rtss-repeat_v4_F_MIP & csp-rtss-repeat_v4_R_MIP)	1	105.6
Template DNA	5

	A	B
	Reagent	Volume
	DNA Template	50 µL
	Ultra II End Prep Reaction Buffer	7 µL
	Ultra II End Prep Enzyme Mix	3 µL
	Total	60µL

	A	B
	Reagent	Volume
	End-prepped DNA	60 µL
	Ligation Buffer (LNB)	25 µL
	NEBNext Quick T4 Ligase	10 µL
	Ligation Adapter (LA)	5 µL
	Total	100 µL

	A	B
	Reagent	Volume
	Flow Cell Flush (FCF)	1170 µL
	Flow Cell Tether (FCT)	30 µL
	Total	1200 µL

	A	B
	Reagent	Volume
	Sequencing Buffer (SB)	100 µL
	Library Beads (LIB)	68 µL
	DNA Library	32 µL
	Total	200 µL

	A	B
	Reagent	Volume
	Wash Diluent (DIL)	398 µL
	Wash Mix (WMX)	2 µL


Well Position	Name	Sequence
A01	hybrid_dual_rev_001	CAAGCAGAAGACGGCATACGAGATGTTAAGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A02	hybrid_dual_rev_002	CAAGCAGAAGACGGCATACGAGATTCTAAGTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A03	hybrid_dual_rev_003	CAAGCAGAAGACGGCATACGAGATTGTACATTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A04	hybrid_dual_rev_004	CAAGCAGAAGACGGCATACGAGATAATACCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A05	hybrid_dual_rev_005	CAAGCAGAAGACGGCATACGAGATTGTCTATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A06	hybrid_dual_rev_006	CAAGCAGAAGACGGCATACGAGATTCACTGTTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A07	hybrid_dual_rev_007	CAAGCAGAAGACGGCATACGAGATTTGTATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A08	hybrid_dual_rev_008	CAAGCAGAAGACGGCATACGAGATAGACTAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A09	hybrid_dual_rev_009	CAAGCAGAAGACGGCATACGAGATATTGTCAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A10	hybrid_dual_rev_010	CAAGCAGAAGACGGCATACGAGATGTTCTACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A11	hybrid_dual_rev_011	CAAGCAGAAGACGGCATACGAGATTTCATTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
A12	hybrid_dual_rev_012	CAAGCAGAAGACGGCATACGAGATAAGCGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B01	hybrid_dual_rev_013	CAAGCAGAAGACGGCATACGAGATCGTAGGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B02	hybrid_dual_rev_014	CAAGCAGAAGACGGCATACGAGATAGTTAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B03	hybrid_dual_rev_015	CAAGCAGAAGACGGCATACGAGATGAACGTCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B04	hybrid_dual_rev_016	CAAGCAGAAGACGGCATACGAGATAGAACACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B05	hybrid_dual_rev_017	CAAGCAGAAGACGGCATACGAGATTGTTGGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B06	hybrid_dual_rev_018	CAAGCAGAAGACGGCATACGAGATGGCTAATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B07	hybrid_dual_rev_019	CAAGCAGAAGACGGCATACGAGATGCTAGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B08	hybrid_dual_rev_020	CAAGCAGAAGACGGCATACGAGATTGTACTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B09	hybrid_dual_rev_021	CAAGCAGAAGACGGCATACGAGATAGTTACACGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B10	hybrid_dual_rev_022	CAAGCAGAAGACGGCATACGAGATGTACACGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B11	hybrid_dual_rev_023	CAAGCAGAAGACGGCATACGAGATACTGTAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
B12	hybrid_dual_rev_024	CAAGCAGAAGACGGCATACGAGATGATCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C01	hybrid_dual_rev_025	CAAGCAGAAGACGGCATACGAGATTGCTAACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C02	hybrid_dual_rev_026	CAAGCAGAAGACGGCATACGAGATTAATGCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C03	hybrid_dual_rev_027	CAAGCAGAAGACGGCATACGAGATCTTGTTGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C04	hybrid_dual_rev_028	CAAGCAGAAGACGGCATACGAGATGATACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C05	hybrid_dual_rev_029	CAAGCAGAAGACGGCATACGAGATATACAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C06	hybrid_dual_rev_030	CAAGCAGAAGACGGCATACGAGATCTGGATAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C07	hybrid_dual_rev_031	CAAGCAGAAGACGGCATACGAGATTCATCAGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C08	hybrid_dual_rev_032	CAAGCAGAAGACGGCATACGAGATTTATACCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C09	hybrid_dual_rev_033	CAAGCAGAAGACGGCATACGAGATGCTGATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C10	hybrid_dual_rev_034	CAAGCAGAAGACGGCATACGAGATATAAGGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C11	hybrid_dual_rev_035	CAAGCAGAAGACGGCATACGAGATAAGACTAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
C12	hybrid_dual_rev_036	CAAGCAGAAGACGGCATACGAGATTAGCAACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D01	hybrid_dual_rev_037	CAAGCAGAAGACGGCATACGAGATCAAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT
D02	hybrid_dual_rev_038	CAAGCAGAAGACGGCATACGAGATTCATGATGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT