Hybridization Capture for Targeted NGS
User Manual
Version 5.03

For

June 2023

myBaits Custom DNA-Seq
myBaits Custom RNA-Seq
myBaits Expert (excluding Wheat Exome and Human Affinities)

Access additional manuals at arborbiosci.com/mybaits-manual

FOR RESEARCH USE ONLY. Not intended for diagnostic use.

© Daicel Arbor Biosciences
5840 Interface Drive, Suite 101
Ann Arbor, MI 48103 (USA)+1 (734) 998-0751
info@arbor.daicel.com

MYBAITS is a registered trademark of Biodiscovery, LLC dba Arbor Biosciences. DAICEL ARBOR BIOSCIENCES is a registered trademark of Daicel Corporation. ION
TORRENT is a registered trademark of Life Technologies Corporation. DYNABEADS is a registered trademark of Thermo Fisher Scientific. MYONE is a trademark of
Thermo Fisher Scientific. NEXTERA, ILLUMINA, and TRUSEQ are registered trademarks of Illumina, Inc. KAPA is a registered trademark of Roche Molecular Systems, Inc.
IDT and XGEN are registered trademarks of Integrated DNA Technologies, Inc. PACBIO is a registered trademark of Pacific Biosciences of California, Inc. OXFORDNANOPORE TECHNOLOGIES is a registered trademark of Oxford Nanopore Technologies, Ltd.

INTRODUCTION
myBaits® is an in-solution NGS library target enrichment system, compatible with Illumina®, Ion Torrent®,
and essentially any linear and amplifiable sequencing library. We use a versatile nucleic acid synthesis
technology to produce biotinylated RNA “baits” that are complementary to your sequence targets. Baits andother reagents for NGS target enrichment are supplied with your myBaits kit. After enrichment with myBaits,
libraries may then be sequenced on the aforementioned platforms, or further prepared for PacBio® or Oxford
Nanopore Technologies® sequencing.

Procedure Overview
1. Amplified sequencing library, adapter
blockers, and other hybridization reagentsare combined.

2. Libraries are denatured, allowing blockers
to hybridize to adapters. Baits are thenintroduced and hybridized to targets forseveral hours.

3. Bait-target hybrids are bound to
streptavidin-coated magnetic beads andpulled out of suspension with a magnet.

4. Most non-target DNA is washed away. The
remaining library is then amplified andeither taken directly to sequencing or
further treated.

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Daicel Arbor Biosciences products compatible with this manualThis manual is compatible with current generation myBaits Custom DNA-Seq, Custom RNA-Seq, and Expert
kits, but not Custom Methyl-Seq, Expert Wheat Exome, or Expert Human Affinities kits. Visitarborbiosci.com/mybaits-manual to download other manuals, or email techsupport@arbor.daicel.com forassistance. See Page 4 to select the correct protocol within this manual to follow for your application.

Changes since myBaits manual version 5.02●

Reagent cap colors are updated (contents are unchanged); minor text changes

Changes since myBaits manual version 4●●●

Three distinct myBaits protocols are included for different applications (see Page 4)
Block X replaces Block A and typically provides improved specificity
Buffer E is now supplied for post-cleanup bead resuspension

Note for returning users: Version 5 reagent chemistry typically provides significantly higher target
specificity (= percentage of on-target reads) compared to version 4 and earlier kits.

Kit components and stability
Box

Box #1
Store at 4˚CBox #2
Store at -20˚C

Box #3
Store at -80˚C

Reagent

Cap color

Volume per kit

Lot# 23XXXX

Lot# 22XXXX

8 Reaction

16 Reaction

48+ Reaction

Hyb N
Hyb S
Beads
Binding Buffer
Wash Buffer
Hyb D
Hyb R
Block C
Block O
Block X
Buffer E

Red
Blue
Clear
Clear
Clear
Yellow
PurpleGreenBlue
Orange
Clear

Red
Teal
Clear
Clear
Clear
Yellow
PurpleDark GreenBlue
OrangeLight Green

400 µL
1500 µL
550 µL
12 mL
20 mL
140 µL
50 µL
50 µL
90 µL
5 µL
550 µL400 µL
1500 µL
550 µL
12 mL
20 mL
140 µL
50 µL
50 µL
90 µL
9 µL
550 µL

1000 µL
1500 µL
1600 µL
36 mL
60 mL
400 µL150 µL130 µL270 µL
27 µL
1600 µL

Baits

Clear

White

50 µL per 8 reactions

myBaits kits are shipped at ambient temperature and are stable during transit. If the kit boxes are stored
upon arrival at the temperatures listed above, the reagents expire one year from the date they were received.
To minimize freeze-thaw cycles, it is strongly recommended that sub-aliquots of Baits are made in reaction
sizes appropriate for your experiment plans.

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IMPORTANT
READ BEFORE PROCEEDINGCHOOSE A PROTOCOLThis manual includes three separate myBaits protocols. Please select from the list below depending on thenature of your sequencing libraries. If you are unsure which protocol to follow, email
techsupport@arbor.daicel.com for consultation with one of our NGS scientists.
Note that these are suggested starting points. Identification of optimal parameters for enrichment with a
specific bait set and library will require testing and evaluation. These protocols have worked well across
many bait set and library combinations.

Standard

Begins on Page 5

For most targeted sequencing applications
Recommended when enriching NGS libraries that:- derive from high-quality genomic DNA, with target inserts of 80-1,000 bp in length,- contain a mean target sequence GC content of >25%,- are free of substantial (>50%) contamination from non-target organisms, and- do not have extensive base substitutions (from e.g. deamination or bisulfite conversion)

High Sensitivity

Begins on Page 17

For high background contamination, ancient DNA, high bait-target divergence, OR short target sequences
Recommended when enriching NGS libraries that have ANY of the following characteristics:- derive from DNA samples heavily contaminated with exogenous DNA (e.g. pathogentargets in host DNA background, ancient DNA, environmental DNA),- contain mostly target sequences shorter than 80 bp,- contain a mean target sequence GC content of <25%, or- contain targets that are on average ≥25% divergent from the bait sequences

Long Insert

Begins on Page 29

For target enrichment of NGS libraries containing inserts 1-10 kilobase pairs in length
Recommended when enriching NGS libraries that:- are dominated by inserts longer than 1,000 bp, and- can be amplified with universal adapter primers prior to platform-specific library prep (e.g.
PacBio or Nanopore library prep)

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Standard Protocol
For most targeted sequencing applications

CONTENTSREQUIREMENTS AND RECOMMENDATIONS
Input library
Blocking oligos
Equipment and reagents

6
6
7

PROCEDURE Part 1: Hybridization Setup
S1.1
Choose a hybridization temperature
S1.2
Prepare materials
S1.3
Hybridization Mix setup
S1.4
Blockers Mix setup
S1.5
Reaction assembly

8
8
9
9
10

PROCEDURE Part 2: Bind and Wash
S2.1
Choose a wash temperature
S2.2
Prepare materials
S2.3
Wash Buffer X preparation
S2.4
Bead preparation
S2.5
Binding beads and hybrids
S2.6
Bead washing

11
11
12
12
12
12

PROCEDURE Part 3: Library Resuspension and Amplification
S3.1
Prepare materials
S3.2
Enriched library recovery
S3.3
Library amplification

13
13
13

APPENDIX
SA1
Troubleshooting
SA2
myBaits Standard Protocol Quick Guide

15
16

5

REQUIREMENTS AND RECOMMENDATIONS
Input library
TYPE
Use myBaits with PCR-amplified and amplifiable NGS libraries, including Illumina
TruSeq®-style, Illumina Nextera® Flex-style, Ion Torrent, or other libraries with universal adapter primingsites. It is NOT recommended to use myBaits with PCR-free libraries; additionally, myBaits are incompatible
with libraries made using original Nextera or Nextera XT library preparation kits, or any library typecontaining biotin. Dual-indexed libraries are strongly recommended to reduce the hazard of mis-indexing
induced by PCR jumping events.
VOLUME
Each myBaits target enrichment reaction has space for 7 µL total NGS library volume. Manypools will require concentration to 7 µL with vacuum centrifugation or other means. Complete this inadvance of your myBaits experiments. Libraries should be suspended in nuclease-free buffer or water.
MASS
A wide range of total library mass can be successfully enriched with myBaits (nanograms
to micrograms). However, best results are seen when 100 ng or more total mass is used per enrichment
reaction. It is strongly recommended that library qPCR is used to measure library mass, rather than e.g. dye
assay or electrophoresis. See note below on default recommendations for mass input for library pools.
POOLING
Optimal pooling parameters (both in terms of number of libraries and total mass per library)
will vary between library types and bait sets, and will require trials to identify. However, many configurations
should work well. To minimize variation in capture performance among pooled samples, only pool libraries
based on comparable anticipated bait-genome sequence identity (i.e, taxon), DNA quality, starting DNA
amount, library insert length, and relative target constituent vs. background. Pool equal amounts of eachlibrary. We recommend that when libraries derive from organisms with genome sizes smaller than 5 Gbp,
pool eight libraries, 250 ng each, for a total of 2 µg per enrichment reaction. For genomes 5 Gbp or larger,
pool eight libraries, 1 µg each for a total of 8 µg.

Blocking oligos
When ordering your myBaits kit, please indicate the sequencing library configuration you intend to enrich.
The standard adapter blocking reagent provided with the kit (Block X) is compatible with Illumina
TruSeq-style or Nextera-style libraries with single 6-12 bp or dual 6-12 bp indexing. For different adapter
configurations, we recommend ordering Custom IDT® xGen® Blocking Oligos customized for your NGS
library adapter sequences. At a concentration of 1 μg/μL, any custom adapter-blocking oligos can be usedin lieu of myBaits Block X.

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Equipment required
Item

Notes

50 mL tubes

1 per 44 reactions

Microcentrifuge tubes (1.5, 1.7, or 1.8 mL)

2 per 8 reactions

0.2 mL PCR strips with attached lids

2 per 8 reactions (e.g., VWR Cat# 93001-118)

Pipettors and tips for 0.5 - 500 μL

Multichannel for 20 and 500 μL recommended✝

Thermal cycler with heated lid compatible with 0.2 mL strips

1 or 2Magnetic particle collector for microcentrifuge tubes

1 (e.g., ThermoFisher Cat# 12321D)

Magnetic particle collector for 0.2 mL strips

1 (e.g., Permagen Cat# S500)

Vortex mixer and mini-centrifuge for tubes and strips
Water bath or incubation oven at 65°C
Heat block for microcentrifuge tubes at 60°C✝

Ensure that the thermal cycler and strips allow no more than 4 µL of 30 µL volume evaporation overnight at 65°C

Reagents required
Reagent

Notes

Nuclease-free (“NF”) water

900 µL per reaction

PCR primers to amplify sequencing libraries after capture, e.g.:
Illumina P5: AATGATACGGCGACCACCGA

2.5 uL @ 10 μM per reaction

Illumina P7: CAAGCAGAAGACGGCATACGA

2.5 uL @ 10 μM per reaction

PCR reagents for post-capture amplification

1 per reaction (e.g. Roche Cat# 07958927001)

PCR purification system, e.g., silica columns or SPRI beads

1 cleanup per reaction

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PROCEDURE
PART 1: Hybridization setup
Sequencing libraries are mixed with various blocking nucleic acids, denatured, and then combined with otherhybridization reagents (including baits). These hybridization reactions incubate for several hours to allow
baits to encounter and hybridize with target library molecules.

S1.1 Choose a hybridization temperature (TH)

65°C

62°C

60°C

When bait-target sequence
divergence is expected to beless than 10%

When bait-target sequence
divergence is expected to be
10 to 15%

When bait-target sequence
divergence is expected to be
15 to 25%

S1.2 Prepare materials
Reagents

Equipment

Hyb N and Hyb S from Box 1

Nuclease-free microcentrifuge tubes (×2)

Hyb D and Hyb R from Box 2

0.2 mL strips with attached lids (×1 per 8 reactions)

Block C, Block O, and Block X from Box 2

Pipettors and tips; multichannel for 20 µL recommended

Baits from Box 3 KEEP ON ICE

Vortex mixer and mini-centrifuge for above tube types

Libraries or library pools in 7 µL per reaction

Heat block set to 60°C
Thermal cycler(s); 2 blocks recommended for 24 or more reactions

Program the thermal cycler:
Set lid temperature 5 to 10°C above
each step temperature to minimize
evaporation

Step

Temperature

Time

1
2
3

95°C
TH
TH

5m
5m
∞

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S1.3 Hybridization Mix setup
1. Once the Hyb reagents have thawed, vortex to homogenize and then briefly centrifuge.
Heat Hyb N and Hyb S to 60°C and vortex to dissolve any precipitate present after thawing
2. Assemble the Hybridization Mix in a microcentrifuge (MC) tube, briefly vortex and briefly centrifuge
the contents to collect. The following volumes are already adjusted for pipetting error:
ComponentµL / Reaction

Hyb N

9.25

Hyb D

3.5

Hyb S*

0.5

Hyb R

1.25

Baits

5.5

TOTAL

20

*Cloudiness caused by Hyb S addition will clear after step 3

3. Incubate the Hybridization Mix at 60°C for 10 minutes in the heat block. Vortex occasionally tocollect the condensate. Remove from the heat block and let sit 5 minutes at room temperature.
4. For each capture reaction, aliquot 18.5 µL of Hybridization Mix to a 0.2 mL well/tube.
These reaction aliquots of Hybridization Mix are now referred to as “HYBs”

S1.4 Blockers Mix setup
1. Assemble the Blockers Mix specific for your target taxon/taxa in an appropriately-sized tube andmix by pipetting. The following volumes are already adjusted for pipetting error:
MOST TAXAPLANTSSALMONIDS

Component µL / Reaction

Component µL / Reaction

Component µL / Reaction

Block O

2.5

Block O

5.0

Block O-

Block C

2.5

Block C-

Block C

2.5

Block X

0.5

Block X

0.5

Block X

0.5

NF Water-NF Water-NF Water

2.5

TOTAL

5.5

TOTAL

5.5

TOTAL

5.5

2. For each capture reaction, aliquot 5 µL of Blockers Mix to a 0.2 mL well/tube.
3. Add 7 µL of individual or pooled libraries to each Blockers Mix aliquot and mix by pipetting.
These libraries mixed with Blockers Mix aliquots are now referred to as “LIBs”

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S1.5 Reaction assembly
Double-check the thermal program:
Set lid temperature 5 to 10°C
above each step temperature tominimize evaporation

Step

Temperature

Time

1
2
3

95°C
TH
TH

5m
5m
∞

1. Put the LIBs in the thermal cycler, close
the lid, and start the thermal program.

2. Oncethecyclerreachesthehybridization temperature during step 2,
pause the program, put the HYBs in thethermal cycler, close the lid, and resume
the program.

3. After step 2 of the program is complete,leaving all tubes in the thermal cycler,
pipette 18 µL of each HYB to each LIB.
Use a multichannel pipettor for easierexecution. Gently homogenize by
pipetting up and down 5 times.
4. Dispose of the HYB tubes. Briefly spin
down the LIBs, return to the thermal
cycler, close the lid, and allow thereactions to incubate overnight (16 to 24
hours).

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PART 2: Bind and Wash (“Cleanup”)
Bait-target hybrids are bound to streptavidin-coated magnetic beads, and then most non-target DNA isremoved with several rounds of washing with a warm buffer. This is usually performed the day following
completion of Part 1.

S2.1 Choose a wash temperature (TW - typically identical to TH)

65°C

62°C

60°C

When bait-target sequence
divergence is expected to be
10% or less

When bait-target sequence
divergence is expected to be
10 to 15%

When bait-target sequence
divergence is expected to be
15 to 25%

S2.2 Prepare materials
Start at least 90 minutes before intended hybridization stop time
Reagents
Hyb S (Box 1) *Binding Buffer (Box 1) *Wash Buffer (Box 1) *Beads (Box 1)* Allow these reagents to come to room temperature
before use; warm to 60°C and vortex to dissolve
precipitate if necessary

Nuclease-free (NF) Water (up to 900 µL per reaction)

Equipment
Water bath or incubation oven set to the TW (e.g., 65°C)
Receptacles for 50 mL tubes, 0.2 mL strips and microcentrifuge tubes compatible with above incubation device
Vortex mixer and mini-centrifuge for 0.2 mL strips and MC tubes
Magnetic particle collector(s) (MPC) for above strips and/or tubes
When using only amicrocentrifuge (MC) tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 44 cleanups
Nuclease-free MC tubes, 1 per reaction
Heat block set to the TW
Pipettors and tips for 20 – 500 µL

When using a
0.2 mL tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 68 cleanups
Nuclease-free 0.2 mL PCR strips withindividually-attached lids, 1 vessel per reaction
Thermal cycler set to TW
Pipettors and tips for 20 – 200 µL;
multichannel pipettor strongly recommended

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S2.3 Wash Buffer X preparation
This step generates enough Wash Buffer X for 44 reactions in microcentrifuge (“MC”) tube cleanup format,
and 68 reactions in 0.2 mL cleanup format; scale up or down if needed.
1. Thaw and thoroughly homogenize Wash Buffer and Hyb S prior to aliquoting in order to dissolve any
visible precipitate; warm slightly if necessary.
2. Combine 400 µL Hyb S, 39.6 mL NF water and 10 mL Wash Buffer in a 50 mL tube. Vortex
thoroughly, label “Wash Buffer X.”
Wash Buffer X can be stored at 4°C for 1 month
3. Heat the Wash Buffer X to the TW in the water bath or oven for at least 30 minutes before use.

S2.4 Bead preparation

Prepare beads immediately prior to use

1. For each capture reaction, aliquot 30 µL beads to a microcentrifuge tube.
2. Pellet the beads in the MPC until the suspension is clear (1-2 minutes).
Leaving the tubes on the magnet, remove and discard the supernatant.
3. Add 200 µL Binding Buffer to each bead aliquot. Vortex to resuspend the beads and centrifuge
briefly. Place tube in the MPC and pellet beads; remove and discard the supernatant.
4. Repeat Step 3 above twice for a total of three washes.
5. Resuspend each washed bead aliquot in 70 µL Binding Buffer. If proceeding to washing in 0.2 mL
format, transfer the aliquots to PCR strips.
TIP: Beads can be prepared in 8 (or fewer) reaction batches (240 µL) in a microcentrifuge tube. Multiply allvolumes by the number of reactions in the batch; i.e., for 8 reactions-worth, wash with 1.6 mL and resuspend in
560 µL Binding Buffer, then aliquot 70 µL suspension to individual tubes.

S2.5 Binding beads and hybrids
1. Heat the bead aliquots to the TW (e.g., 65°C) for at least 2 minutes.
2. Transfer each capture reaction to the heated bead aliquots. Mix by pipetting.
3. Incubate the libraries + beads on the heat block or thermal cycler for 5 minutes. Agitate at the 2.5
minute mark by flicking or inverting to keep the beads suspended, followed by briefly centrifuging.

S2.6 Bead washing
1. Pellet the beads with the MPC until the solution is clear. Remove and discard the supernatant.
TIP: This supernatant can be optionally saved as a backup for re-capture with the same or different baits.

2. Add 375 µL (MC tube format) or 180 µL (0.2 mL format) warmed Wash Buffer X to the beads,
remove from the MPC, place on heat block for 15 seconds, and briefly vortex or mix by pipetting.
Briefly centrifuge the mixture.
3. Incubate for 5 minutes at the TW in the heat block or thermal cycler. Agitate at the 2.5 minute markvia gentle vortexing and then briefly centrifuge.
4. Repeat steps 1 through 3 two times for MC tube format (three washes total), or three times for 0.2
mL format (four washes total). After the last wash and pelleting, remove as much liquid as
possible without touching the bead pellet.

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PART 3: Library Resuspension and Amplification
Bead-bound enriched library is resuspended in Buffer E and amplified.

S3.1 Prepare materials
Reagents

Equipment

Buffer E (Box 2)

Tubes appropriate for PCR master mix assembly

PCR primers for amplifying libraries (e.g., P5 and P7)

Tubes or strips for 50 µL PCR amplification

PCR reagents for post-capture amplification

Pipettors and tips capable of 5 – 100 µL volumes

PCR purification system, e.g., silica columns or SPRI beads

Vortex mixer and mini-centrifuge for above tube types

Nuclease-free (NF) Water

Thermal cycler

S3.2 Enriched library recovery
1. Add 30 µL Buffer E to the washed beads and thoroughly resuspend by pipetting.
Then, depending on your library amplification system, choose workflow A or B:
WORKFLOW A: When using KAPA HiFi HotStart or NEB
Ultra II Q5 polymerase systems for amplification

WORKFLOW B: When not using the polymerase
systems for amplification in workflow A

2B.
2A. Proceed directly to section S3.3 using
this bead resuspension as the template inamplification

3B.

Incubate the suspension at 95°C
for 5 minutes
Immediately pellet the beads in the MPC
and collect the supernatant containing
the enriched libraries

S3.3 Library amplification
This is an example post-capture amplification using KAPA HiFi HotStart ReadyMix and Illumina libraries:
1. Assemble the following PCR master mix:
Component

Final ConcentrationµL / Reaction

Sequence

NF Water-

5-

2X KAPA HiFi HotStart Ready Mix

1X

25

-

P5 library primer (at 10 μM)

500 nM

2.5

AATGATACGGCGACCACCGA

P7 library primer (at 10 μM)

500 nM

2.5

CAAGCAGAAGACGGCATACGA

-

15 *

-

50

-

Enriched Library (on- or off-bead)

TOTAL*Remaining bead-bound library can be stored at -20°C for several months.

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S3.3 Library amplification (continued)
2. Cycle the reactions with the following thermal program:
Step

Temperature

Time

1

98°C

2 minutes

2

98°C

20 seconds

3

60°C

30 seconds

4

72°C

45 seconds

5

72°C

5 minutes

6

8°C

∞

×8 to 14cycles**Minimize cycles where possible.
Cycles required to meet molarity
requirements of sequencing
platform may exceed 14.

3. After amplification:- If beads were included in the amplification reaction and you intend to use silica columns
for purification, pellet the beads first and purify only the supernatant.
-

Otherwise, purify the reaction using your preferred PCR cleanup (e.g., silica columns or
SPRI beads).

The enriched libraries are now ready for quantification, quality-assessment, and sequencing.

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APPENDIX
SA1: Troubleshooting
During hybridization, my thermal cycler dropped below the hybridization temperature.
You can expect a lower on-target read proportion and target read complexity for these libraries thanif the temperature remained where intended, but not outright enrichment failure. Shallow preliminary
sequencing will determine whether targets are likely to be retrieved at sufficient coverage within budget.
My enriched and amplified library is not visible on electrophoresis gel or similar.
Successful captures frequently yield a total mass of just a few nanograms even after
re-amplification, which can be difficult to visualize with electrophoresis. This is most common when
capturing especially small targets (<100 bp), or targets that are present at low frequency in the starting
library (like those in degraded/ancient/environmental DNA), or if there is under-reamplification of the librarypost-capture. Often a few more cycles of library amplification will render the captured product sufficiently
high in concentration to view with electrophoresis. Alternatively, determine with library qPCR whether the
library is of sufficient mass for sequencing. If cycling is halted before reaching PCR plateau, the qPCR
product can be visualized with electrophoresis to determine length distribution. Consult with yoursequencing provider for library concentration and volume requirements.
My enriched and amplified library appears significantly longer than my original library, or has two peaks.
This may happen if the libraries are over-amplified and have formed ‘daisy-chains’ or ‘bubbles’ by
experiencing cycles of denature-renature without template extension. These can be reverted to their original
appearance in electrophoresis by applying three PCR cycles using regular library amplification.
I observe a high ratio of PCR duplicates in my enriched library sequence data.
Percent duplicates in sequencing data (i.e. “clonality” or “duplication rate”) increases as you
sequence deeper, and therefore it can only be fairly compared between experiments when the sequencing
depth is normalized before analysis. Evaluate whether you have simply over-sequenced the libraries byplotting raw sequencing reads obtained on the X axis, and unique reads observed on the Y axis. If thiscomplexity curve has plateaued, but you achieved sufficient unique reads, you sequenced more deeply thannecessary. If it has not flattened, or you need to increase the total potential unique read yield of the library,use more DNA per library preparation and/or more library per capture reaction. Avoid diluting baits before
capture. When working with heavily contaminated or damaged DNA target molecules, consider reducing
temperatures used in all steps to improve capture sensitivity. Reducing PCR cycles when possible may also
improve target coverage uniformity and complexity for a given sequencing depth, in some cases having anindirect effect on duplication rate. For more information about library complexity for any NGS application,
we recommend Daley & Smith 2013 (doi: 10.1038/nmeth.2375).

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SA2: myBaits Procedure Quick Guide - Standard Protocol
1.

For each reaction, build the following Mixes; pipetting error is built in:Hybridization Mix

Blockers Mix

Component

μL / Reaction

Component

μL / Reaction

Hyb N

9.25

Block X

0.5

Hyb D

3.5

Block C

2.5✝

Hyb S

0.5

Block O

2.5*

Hyb R

1.25

NF Water

0✝

Baits

5.5

TOTAL

5.5

TOTAL

20

Plants: 0; Salmonids: 2.5*Plants: 5.0; Salmonids: 0✝

2.

After pre-warming the Hybridization mix for 10 minutes @ 60°C, for each reaction, aliquot 18.5 µL of
Hybridization Mix to their own tubes – now “HYBs”.

3.

For each reaction, aliquot 5 µL of Blockers Mix and then add 7 µL of each library – now “LIBs”.

4.

Incubate the LIBs in the thermal cycler for 5 minutes @ 95°C and then drop to the hybridization temperature(e.g., 65°C). Be sure to use a heated lid.

5.

Put the HYBs in the thermal cycler and warm to the hybridization temperature for 5 minutes.

6.

Transfer 18 µL of each HYB to each LIB, mix by pipetting, and incubate for 16-24 hours.

7.

1.5 hours before step 9, prepare Wash Buffer X by combining 400 µL Hyb S, 39.6 mL nuclease-free molecularbiology-grade water and 10 mL Wash Buffer in a 50 mL tube. Vortex thoroughly and warm to the hybridization
temperature for at least 45 minutes.

8.

Prepare 30 µL of beads per reaction by washing three times in 200 µL Binding Buffer. Resuspend the washedbead aliquots in 70 µL Binding Buffer and warm the suspensions to the hybridization temperature for at least 2
minutes.

9.

Combine the warmed beads with the hybridization reactions and incubate for 5 minutes at the hybridization
temperature, agitating at 2.5 minutes to keep beads suspended.

10. Pellet the beads and remove the supernatant. If using microcentrifuge tubes for cleanup, wash the beads threetimes with 375 µL warmed Wash Buffer X, incubating 5 minutes at the hybridization temperature. Wash fourtimes with 180 µL washes if using a 96-well magnetic particle concentrator and 0.2 mL strips/tubes.
11. Resuspend the beads in 30 µL Buffer E and then use 15 µL of this in a 50 µL amplification reaction with KAPA®
HiFi or NEB Ultra II Q5 polymerase systems. If not using these polymerase systems, instead elute the libraryfrom the beads by incubating the suspension for 5 minutes at 95°C, immediately pellet the beads, and then use
15 µL of the supernatant in a 50 µL amplification reaction.
12. Purify the amplification reactions using silica columns or SPRI beads. If using silica columns and beads were
included in the amplification reaction, pellet the beads first and purify only the supernatant.

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High Sensitivity Protocol
For high background contamination, ancient DNA, high bait-target divergence, and/or short insert fragments

CONTENTSREQUIREMENTS AND RECOMMENDATIONS
Input library
Blocking oligos
Special note on two-round enrichment protocols
Equipment and reagents

18
18
19
19

PROCEDURE Part 1: Hybridization Setup
H1.1 Choose a hybridization temperature
H1.2 Prepare materials
H1.3 Hybridization Mix setup
H1.4 Blockers Mix setup
H1.5 Reaction assembly

20
20
21
21
22

PROCEDURE Part 2: Bind and Wash
H2.1 Choose a wash temperature
H2.2 Prepare materials
H2.3 Wash Buffer X preparation
H2.4 Bead preparation
H2.5 Binding beads and hybrids
H2.6 Bead washing

23
23
24
24
24
24

PROCEDURE Part 3: Library Resuspension and Amplification
H3.1 Prepare materials
H3.2 Enriched library recovery
H3.3 Library amplification
H3.4 Perform a second round of enrichment

25
25
25
26

APPENDIX
HA1
Troubleshooting
HA2
myBaits High Sensitivity Protocol Quick Guide

27
28

17

REQUIREMENTS AND RECOMMENDATIONS
Input library
TYPE
Use myBaits with PCR-amplified and amplifiable NGS libraries, including Illumina
TruSeq®-style, Illumina Nextera® Flex-style, Ion Torrent, or other libraries with universal adapter primingsites. It is NOT recommended to use myBaits with PCR-free libraries; additionally, myBaits are incompatible
with libraries made using original Nextera or Nextera XT library preparation kits, or any library typecontaining biotin. Dual-indexed libraries are strongly recommended to reduce the hazard of mis-indexing
induced by PCR jumping events.
VOLUME
Each myBaits target enrichment reaction has space for 7 µL total NGS libraryvolume. Many pools will require concentration to 7 µL with vacuum centrifugation or other means. Perform
this in advance of your myBaits experiments. Libraries should be suspended in nuclease-free buffer orwater.
MASS & POOLING
A wide range of total library mass can be successfully enriched with myBaits(nanograms to micrograms). Optimal pooling parameters (both in terms of number of libraries and total
mass per library) will vary between library types and bait sets and may require trials to identify. However,many configurations should work well.
Non-degraded, non-contaminated DNA libraries (e.g. libraries with bait-target divergence
expected mean >25%): To minimize variation in capture performance among pooled samples, only poollibraries of comparable anticipated bait-genome sequence identity (i.e, taxon), DNA quality, starting DNA
amount, library insert length, and relative target constituent vs. background. Pool equal amounts of eachlibrary. When libraries derived from organisms with genome sizes smaller than 5 Gbp, pool eight libraries,
250 ng each, for a total of 2 µg per enrichment reaction. For genomes 5 Gbp or larger, pool eight libraries, 1µg each for a total of 8 µg.
Degraded and contaminated DNA libraries: Pool as few libraries as possible, and input as much as
possible to capture, up to a total of 12 µg. If pooling is required, make certain that libraries are balanced byendogenous content to ensure each sample contributes the same amount of target content. For example, if
two libraries are to be pooled, and library A has 20% endogenous template and library B has 10%
endogenous template, include twice the mass of library B as library A in the enrichment pool.

Blocking oligos
When ordering your myBaits kit, please indicate the sequencing library configuration you intend to enrich.
The standard adapter blocking reagent provided with the kit (Block X) is compatible with Illumina
TruSeq-style or Nextera-style libraries with single 6-12 bp or dual 6-12 bp indexing. For different adapter
configurations, we recommend ordering Custom IDT® xGen® Blocking Oligos customized for your NGS

myBaits® - Hybridization Capture for Targeted NGS - Manual v.5.03 - High Sensitivity Protocol June 202318

library adapter sequences. At a concentration of 1 μg/μL, any custom adapter-blocking oligos can be usedin lieu of myBaits Block X.

Special note on two-round enrichment protocols:
The High Sensitivity protocol includes two rounds of enrichment. This may require that you purchaseadditional myBaits Reagents (without Baits). For example, if you plan to use all 16 reactions of a myBaits kit
in the High Sensitivity protocol, you will need to have both the 16 reaction kit as well as at least 16
additional reactions-worth of myBaits Reagents. These can be purchased in 16 or 48 reaction kits, catalog
numbers 300016 and 300048, respectively.

Equipment required
Item

Notes

50 mL tubes

1 per 44 reactions

Microcentrifuge tubes (1.5, 1.7, or 1.8 mL)

2 per 8 reactions

0.2 mL PCR strips with attached lids

2 per 8 reactions (e.g., VWR Cat# 93001-118)

Pipettors and tips for 0.5 - 500 μL

Multichannel for 20 and 500 μL recommended✝

Thermal cycler with heated lid compatible with 0.2 mL strips

1 or 2Magnetic particle collector for microcentrifuge tubes

1 (e.g., ThermoFisher Cat# 12321D)

Magnetic particle collector for 0.2 mL strips

1 (e.g., Permagen Cat# S500)

Vortex mixer and mini-centrifuge for tubes and strips
Water bath or incubation oven at 65°C
Heat block for microcentrifuge tubes at 60°C✝

Ensure that the thermal cycler and strips allow no more than 4 µL of 30 µL volume evaporation overnight at 65°C

Reagents required
Reagent

Notes

Nuclease-free (“NF”) water

900 µL per reaction

PCR primers to amplify sequencing libraries after capture, e.g.:
Illumina P5: AATGATACGGCGACCACCGA

2.5 uL @ 10 μM per reaction

Illumina P7: CAAGCAGAAGACGGCATACGA

2.5 uL @ 10 μM per reaction

PCR reagents for post-capture amplification

1 per reaction (e.g. Roche Cat# 07958927001)

PCR purification system, e.g., silica columns or SPRI beads

1 cleanup per reaction

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PROCEDURE
PART 1: Hybridization setup
Sequencing libraries are mixed with various blocking nucleic acids, denatured, and then combined with otherhybridization reagents (including baits). These hybridization reactions incubate for several hours to allow
baits to encounter and hybridize with target library molecules.

H1.1 Choose a hybridization temperature (TH)

63°C

60°C

55°C

For DNA libraries with low GC
For libraries with expected
For libraries with insert length
content or with expected bait-target bait-target sequence divergence distributions mostly shorter thandivergence of less than 15%
of 15 to 25%
the myBaits probe length, or
expected bait-target sequence
divergence higher than 25%

H1.2 Prepare materials
Reagents

Equipment

Nuclease-free Water (“H2O”)

Nuclease-free microcentrifuge tubes (×2)

Hyb N and Hyb S from Box 1

0.2 mL strips with attached lids (×1 per 8 reactions)Hyb D and Hyb R from Box 2

Pipettors and tips; multichannel for 20 µL recommended

Block C, Block O, and Block X from Box 2

Vortex mixer and mini-centrifuge for above tube types

Baits from Box 3 KEEP ON ICEHeat block set to 60°C

Libraries or library pools in 7 µL per reaction

Thermal cycler(s); 2 blocks recommended for 24 or more reactions

Program the thermal cycler:
Set lid temperature 5 to 10°C above
each step temperature to minimize
evaporation

Step

Temperature

Time

1
2
3

95°C
TH
TH

5m
5m
∞

H1.3 Hybridization Mix setup
1. Once the Hyb reagents have thawed, vortex them to homogenize and then briefly centrifuge.
Heat Hyb N and Hyb S to 60°C and vortex to dissolve any precipitate present after thawing

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2. Assemble the Hybridization Mix in a microcentrifuge (MC) tube, briefly vortex and briefly centrifuge
to collect the sample. The following volumes are already adjusted for pipetting error:
ComponentµL / Reaction

Hyb N

9.25

Hyb D

3.5

Hyb S*

0.5

Hyb R

1.25

*Cloudiness caused by Hyb S addition will clear after step 3

H2O (round 1 / round 2)

1.1 / 4.4

First enrichment round: 1.1 μL Second round: 4.4 μL

Baits (round 1 / round 2)

4.4 / 1.1

First enrichment round: 4.4 μL Second round: 1.1 μL

TOTAL

20

3. Incubate the Hybridization Mix at 60°C for 10 minutes in the heat block. Vortex occasionally tocollect condensate. Remove from the heat block and let sit 5 minutes before proceeding.
4. For each capture reaction, aliquot 18.5 µL of Hybridization Mix to a 0.2 mL well/tube.
These reaction aliquots of Hybridization Mix are now referred to as “HYBs”

H1.4 Blockers Mix setup
1. Assemble the Blockers Mix specific for your target taxon/taxa in an appropriately-sized tube andmix by pipetting. The following volumes are already adjusted for pipetting error:
MOST TAXAPLANTSSALMONIDS

Component µL / Reaction

Component µL / Reaction

Component µL / Reaction

Block O

2.5

Block O

5.0

Block O-

Block C

2.5

Block C-

Block C

2.5

Block X

0.5

Block X

0.5

Block X

0.5

NF Water-NF Water-NF Water

2.5

TOTAL

5.5

TOTAL

5.5

TOTAL

5.5

2. For each capture reaction, aliquot 5 µL of Blockers Mix to a 0.2 mL well/tube.
3. Add 7 µL of individual or pooled libraries to each Blockers Mix aliquot and mix by pipetting.
These libraries mixed with Blockers Mix aliquots are now referred to as “LIBs”

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H1.5 Reaction assembly
Double-check the thermal program:
Set lid temperature 5 to 10°C
above each step temperature tominimize evaporation

Step

Temperature

Time

1
2
3

95°C
TH
TH

5m
5m
∞

1. Put the LIBs in the thermal cycler, close
the lid, and start the thermal program.

2. Oncethecyclerreachesthehybridization temperature during step 2,
pause the program, put the HYBs in thethermal cycler, close the lid, and resume
the program.

3. After step 2 of the program is complete,leaving all tubes in the thermal cycler,
pipette 18 µL of each HYB to each LIB.
Use a multichannel pipettor for easierexecution. Gently homogenize by
pipetting up and down 5 times.
4. Dispose of the HYB tubes. Briefly spin
down the LIBs, return to the thermal
cycler, close the lid, and allow thereactions to incubate overnight (16 to 24
hours).

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PART 2: Bind and Wash (“Cleanup”)
Bait-target hybrids are bound to streptavidin-coated magnetic beads, and then most non-target DNA isremoved with several rounds of washing with a warm buffer. This is usually performed the day following
completion of Part 1.

H2.1 Choose a wash temperature (TW - typically identical to TH)

63°C

60°C

55°C

When bait-target sequence
divergence is expected to be
15% or less

When bait-target sequence
divergence is expected to be
15 to 25%

When bait-target sequence
divergence is expected to behigher than 25%

H2.2 Prepare materials
Start at least 90 minutes before intended hybridization stop time
Reagents
Hyb S (Box 1) *Binding Buffer (Box 1) *Wash Buffer (Box 1) *Beads (Box 1)* Allow these reagents to come to room temperature
before use; warm to 60°C and vortex to dissolve
precipitate if necessary

Nuclease-free (NF) Water (up to 900 µL per reaction)

Equipment
Water bath or incubation oven set to the TW (e.g., 65°C)
Receptacles for 50 mL tubes, 0.2 mL strips and microcentrifuge tubes compatible with above incubation device
Vortex mixer and mini-centrifuge for 0.2 mL strips and MC tubes
Magnetic particle collector(s) (MPC) for above strips and/or tubes
When using only amicrocentrifuge (MC) tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 44 cleanups
Nuclease-free MC tubes, 1 per reaction
Heat block set to the TW
Pipettors and tips for 20 – 500 µL

When using a
0.2 mL tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 68 cleanups
Nuclease-free 0.2 mL PCR strips withindividually-attached lids, 1 vessel per reaction
Thermal cycler set to TW
Pipettors and tips for 20 – 200 µL;
multichannel pipettor strongly recommended

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H2.3 Wash Buffer X preparation
This step generates enough Wash Buffer X for 44 reactions in microcentrifuge (“MC”) tube cleanup format,
and 68 reactions in 0.2 mL cleanup format; scale up or down if needed.
1. Thaw and thoroughly homogenize Wash Buffer and Hyb S prior to aliquoting in order to dissolve any
visible precipitate; warm slightly if necessary.
2. Combine 400 µL Hyb S, 39.6 mL NF water and 10 mL Wash Buffer in a 50 mL tube. Vortex
thoroughly, label “Wash Buffer X.”
Wash Buffer X can be stored at 4°C for 1 month
3. Heat the Wash Buffer X to the TW in the water bath or oven for at least 30 minutes before use.

H2.4 Bead preparation

Prepare beads immediately prior to use

1. For each capture reaction, aliquot 30 µL beads to a microcentrifuge tube.
2. Pellet the beads in the MPC until the suspension is clear (1-2 minutes).
Leaving the tubes on the magnet, remove and discard the supernatant.
3. Add 200 µL Binding Buffer to each bead aliquot. Vortex to resuspend the beads and centrifuge
briefly. Place tube in the MPC and pellet beads; remove and discard the supernatant.
4. Repeat Step 3 above twice for a total of three washes.
5. Resuspend each washed bead aliquot in 70 µL Binding Buffer. If proceeding to washing in 0.2 mL
format, transfer the aliquots to PCR strips.
TIP: Beads can be prepared in 8 (or fewer) reaction batches (240 µL) in a microcentrifuge tube. Multiply allvolumes by the number of reactions in the batch; i.e., for 8 reactions-worth, wash with 1.6 mL and resuspend in
560 µL Binding Buffer, then aliquot 70 µL suspension to individual tubes.

H2.5 Binding beads and hybrids
1. Heat the bead aliquots to the TW (e.g., 63°C) for at least 2 minutes.
2. Transfer each capture reaction to the heated bead aliquots. Mix by pipetting.
3. Incubate the libraries + beads on the heat block or thermal cycler for 5 minutes. Agitate at the 2.5
minute mark by flicking or inverting to keep the beads suspended, followed by briefly centrifuging.

H2.6 Bead washing
1. Pellet the beads with the MPC until the solution is clear. Remove and discard the supernatant.
TIP: This supernatant can be optionally saved as a backup for re-capture with the same or different baits.

2. Add 375 µL (MC tube format) or 180 µL (0.2 mL format) warmed Wash Buffer X to the beads,
remove from the MPC, place on heat block for 15 seconds, and briefly vortex or mix by pipetting.
Briefly centrifuge to collect.
3. Incubate for 5 minutes at the TW in the heat block or thermal cycler. Agitate at the 2.5 minute markvia gentle vortexing and briefly centrifuge.
4. Repeat steps 1 through 3 two times for MC tube format (three washes total), or three times for 0.2
mL format (four washes total). After the last wash and pelleting, remove as much fluid as possible
without touching the bead pellet.

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PART 3: Library Resuspension and Amplification
Bead-bound enriched library is resuspended in Buffer E and amplified.

H3.1 Prepare materials
Reagents

Equipment

Buffer E (Box 2)

Tubes appropriate for PCR master mix assembly

PCR primers for amplifying libraries (e.g., P5 and P7)

Tubes or strips for 50 µL PCR amplification

PCR reagents for post-capture amplification

Pipettors and tips capable of 5 – 100 µL volumes

PCR purification system, e.g., silica columns or SPRI beads

Vortex mixer and mini-centrifuge for above tube types

Nuclease-free (NF) Water

Thermal cycler

H3.2 Enriched library recovery
1. Add 30 µL Buffer E to the washed beads and thoroughly resuspend by pipetting.
Then, depending on your library amplification system, choose workflow A or B:
WORKFLOW A: When using KAPA HiFi HotStart or NEB
Ultra II Q5 polymerase systems for amplification

WORKFLOW B: When not using the polymerase
systems for amplification in workflow A

2B.
2A. Proceed directly to section H3.3 using
this bead resuspension as template inamplification

3B.

Incubate the suspension at 95°C
for 5 minutes
Immediately pellet the beads in the MPC
and collect the supernatant containing
the enriched libraries

H3.3 Library amplification
This is an example post-capture amplification using KAPA HiFi HotStart ReadyMix and Illumina libraries.
If this is the first time performing step H3.3, generate two of the following reactions per enrichment
reaction (each with 15 µL enriched library as template). Otherwise, generate only one:
1. Assemble the following PCR master mix:
Component
NF Water
2X KAPA HiFi HotStart Ready Mix
P5 library primer (at 10 μM)
P7 library primer (at 10 μM)
Enriched Library (on- or off-bead)

Final ConcentrationµL / Reaction

Sequence

1X
500 nM
500 nM
-

5
25
2.5
2.5
15 *

AATGATACGGCGACCACCGACAAGCAGAAGACGGCATACGA

TOTAL
50
*Remaining bead-bound library can be stored at -20°C for several months.

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25

H3.3 Library amplification (continued)
2. Cycle the reactions with the following thermal program:
Step

Temperature

Time

1

98°C

2 minutes

2

98°C

20 seconds

3

60°C

30 seconds

4

72°C

45 seconds

5

72°C

5 minutes

6

8°C

∞

×14 or 8

cycles**First round of enrichment: 14
Second round of enrichment: 8

3. After amplification:- If beads were included in the amplification reaction and you intend to use silica columns
for purification, pellet the beads first and purify only the supernatant.
-

Otherwise, purify the reaction using your preferred PCR cleanup (e.g., silica columns or
SPRI beads).

H3.4 Perform a second round of enrichment
If this is the end of the first time through step H3.3 (one of two):
1. Combine both purified amplification reactions generated above and concentrate to 7 µL.
2. Repeat steps H1.1 through H3.3 using this once-enriched template as input.
If this is the end of your second time through step H3.3 (two of two), the enriched libraries are now readyfor quantification, quality-assessment, and sequencing.

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APPENDIX
HA1: Troubleshooting
During hybridization, my thermal cycler dropped below the hybridization temperature
You can expect a lower on-target read proportion and target read complexity for these libraries thanif the temperature had remained where intended, but not outright enrichment failure. Shallow preliminary
sequencing will determine whether targets are likely to be retrieved at sufficient coverage within budget.
My enriched and amplified library is not visible on electrophoresis gel or similar
Successful captures frequently yield a total mass of just a few nanograms even after
re-amplification, which can be difficult to visualize with electrophoresis. This is most common when
capturing especially small targets (<100 bp), or targets that are present at low frequency in the starting
library (like those in degraded/ancient/environmental DNA), or if there is under-reamplification of the librarypost-capture. Often a few more cycles of library amplification will render the captured product sufficiently
high in concentration to view with electrophoresis. Alternatively, determine with library qPCR whether the
library is of sufficient mass for sequencing. If cycling is halted before reaching PCR plateau, the qPCR
product can be visualized with electrophoresis to determine length distribution. Consult with yoursequencing provider for library concentration and volume requirements.
My enriched and amplified library appears significantly longer than my original library, or has two peaks.
This may happen if the libraries are over-amplified and have formed ‘daisy-chains’ or ‘bubbles’ by
experiencing cycles of denature-renature without template extension. These can be reverted to their original
appearance in electrophoresis by applying three PCR cycles using regular library amplification.
I observe a high ratio of PCR duplicates in my enriched library sequence data.
Percent duplicates in sequencing data (i.e. “clonality” or “duplication rate”) increases as you
sequence deeper, and therefore it can only be fairly compared between experiments when the sequencing
depth is normalized before analysis. Evaluate whether you have simply over-sequenced the libraries byplotting raw sequencing reads obtained on the X axis, and unique reads observed on the Y axis. If thiscomplexity curve has plateaued, but you achieved sufficient unique reads, you sequenced more deeply thannecessary. If it has not flattened, or you need to increase the total potential unique read yield of the library,use more DNA per library preparation and/or more library per capture reaction. Avoid diluting baits before
capture. When working with heavily contaminated or damaged DNA target molecules, consider reducing
temperatures used in all steps to improve capture sensitivity. Reducing PCR cycles when possible may also
improve target coverage uniformity and complexity for a given sequencing depth, in some cases having anindirect effect on duplication rate. For more information about library complexity for any NGS application,
we recommend Daley & Smith 2013 (doi: 10.1038/nmeth.2375).

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HA2: myBaits Procedure Quick Guide - High Sensitivity Protocol
1.

For each reaction, build the following Mixes; pipetting error is built in:Hybridization Mix

Blockers Mix

Component

μL / Reaction

Component

μL / Reaction

Hyb N

9.25

Block X

0.5

Hyb D

3.5

Block C

2.5✝

Hyb S

0.5

Block O

2.5*

Hyb R

1.25

NF Water0✝

Baits (round 1 / 2)

4.4 / 1.1

TOTAL

5.5

H2O (round 1 / 2)

1.1 / 4.4

TOTAL

20

Plants: 0; Salmonids: 2.5*Plants: 5.0; Salmonids: 0✝

2.

After pre-warming the Hybridization mix for 10 minutes @ 60°C, for each reaction, aliquot 18.5 µL of
Hybridization Mix to their own tubes – now “HYBs”.

3.

For each reaction, aliquot 5 µL of Blockers Mix and then add 7 µL of each library – now “LIBs”.

4.

Incubate the LIBs in the thermal cycler for 5 minutes @ 95°C and then drop to the hybridization temperature(e.g., 63°C). Be sure to use a heated lid.

5.

Put the HYBs in the thermal cycler and warm to the hybridization temperature for 5 minutes.

6.

Transfer 18 µL of each HYB to each LIB, mix by pipetting, and incubate for 16-24 hours.

7.

1.5 hours before step 9, prepare Wash Buffer X by combining 400 µL Hyb S, 39.6 mL nuclease-free molecularbiology-grade water and 10 mL Wash Buffer in a 50 mL tube. Vortex thoroughly and warm to the hybridization
temperature for at least 45 minutes.

8.

Prepare 30 µL of beads per reaction by washing three times in 200 µL Binding Buffer. Resuspend washed beadaliquots in 70 µL Binding Buffer and warm the suspensions to the hybridization temperature for at least 2
minutes.

9.

Combine the warmed beads with the hybridization reactions and incubate for 5 minutes at the hybridization
temperature, agitating at 2.5 minutes to keep beads suspended.

10. Pellet the beads and remove the supernatant. If using microcentrifuge tubes for cleanup, wash the beads threetimes with 375 µL warmed Wash Buffer X, incubating 5 minutes at the hybridization temperature. Wash fourtimes with 180 µL washes if using a 96-well magnetic particle concentrator and 0.2 mL strips/tubes.
11. Resuspend the beads in 30 µL Buffer E and then use 15 µL of this in a 50 µL amplification reaction with KAPA®
HiFi or NEB Ultra II Q5 polymerase systems. If not using these polymerase systems, instead elute the libraryfrom the beads by incubating the suspension for 5 minutes at 95°C, immediately pellet the beads, and then use
15 µL of the supernatant in a 50 µL amplification reaction. Do two amplifications if this is the first round ofenrichment; do one if this is the second and last round of enrichment.
12. Purify the amplification reactions using silica columns or SPRI beads. If using silica columns and beads were
included in the amplification reaction, pellet the beads first and purify only the supernatant.
13. If this is the end of your first round of enrichment: combine both post-capture amplifications and concentrate to
7 uL, then repeat steps 1-12. Otherwise, the enriched libraries are ready for QC and sequencing.

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Long Insert Protocol
For target enrichment of NGS libraries containing inserts 1-10 kilobase pairs in length

CONTENTSREQUIREMENTS AND RECOMMENDATIONS
Input library
Blocking oligos
Special note for handling long-insert libraries
Equipment and reagents

30
30
30
31

PROCEDURE Part 1: Hybridization Setup
L1.1
Choose a hybridization temperature
L1.2
Prepare materials
L1.3
Hybridization Reaction Setup

32
32
33

PROCEDURE Part 2: Bind and Wash
L2.1
Choose a wash temperature
L2.2
Prepare materials
L2.3
Wash Buffer X preparation
L2.4
Bead preparation
L2.5
Binding beads and hybrids
L2.6
Bead washing

34
34
35
35
35
35

PROCEDURE Part 3: Library Resuspension and Amplification
L3.1
Prepare materials
L3.2
Enriched library recovery
L3.3
Library amplification

36
36
36

APPENDIX
LA1
Troubleshooting
LA2
myBaits Long Insert Protocol Quick Guide

38
39

29

REQUIREMENTS AND RECOMMENDATIONS
Input library
TYPE
Use myBaits with PCR-amplified and amplifiable NGS libraries with universal adapter
priming sites. For long insert library preparation, we recommend the procedure described in Witek et al.
2016 (doi: 10.1038/protex.2016.027). It is NOT recommended to use myBaits with PCR-free libraries;
additionally, myBaits are incompatible with libraries made using original Nextera or Nextera XT library
preparation kits, or any library type containing biotin. Dual-indexed libraries are strongly recommended toreduce the hazard of mis-indexing induced by PCR jumping events.
VOLUME
Each myBaits target enrichment reaction has space for 7 µL total NGS library volume. Manypools will require concentration to 7 µL with vacuum centrifugation or other means. Complete this inadvance of your myBaits experiments. Libraries should be suspended in nuclease-free buffer or water.
MASS
For long insert capture, we recommend using 250 ng total library per enrichment reaction,
as quantified with intercalating dye assay (e.g. Qubit).
POOLING
Optimal pooling parameters (both in terms of number of libraries and total mass per library)
will vary between library types and bait sets, and will require trials to identify. However, many configurations
should work well. To minimize variation in capture performance among pooled samples, only pool libraries
of comparable anticipated bait-genome sequence identity (i.e, taxon), starting DNA amount, library insert
length, and relative target constituent vs. background. Pool equal amounts of each library. For long insert
capture, by default we recommend pooling three libraries, 83 ng each, for a total of ~250 ng per enrichment
reaction.

Blocking oligos
When ordering your myBaits kit, please indicate the sequencing library configuration you intend to enrich.
The standard adapter blocking reagent provided with the kit (Block X) is compatible with Illumina
TruSeq-style or Nextera-style libraries with single 6-12 bp or dual 6-12 bp indexing. For different adapter
configurations, we recommend ordering Custom IDT® xGen® Blocking Oligos customized for your NGS
library adapter sequences. At a concentration of 1 μg/μL, any custom adapter-blocking oligos can be usedin lieu of myBaits Block X.

Special note for handling long-insert libraries
Use gentle repeated pipetting rather than vortexing to homogenize solutions that contain both beads(MyOne C1 or SPRI) and long insert library. This helps reduce potential mechanical shearing.

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30

Equipment required
Item

Notes

50 mL tubes

1 per 44 reactions

Microcentrifuge tubes (1.5, 1.7, or 1.8 mL)

2 per 8 reactions

0.2 mL PCR strips with attached lids

2 per 8 reactions (e.g., VWR Cat# 93001-118)

Pipettors and tips for 0.5 - 500 μL

Multichannel for 20 and 500 μL recommended✝

Thermal cycler with heated lid compatible with 0.2 mL strips

1 or 2Magnetic particle collector for microcentrifuge tubes

1 (e.g., ThermoFisher Cat# 12321D)

Magnetic particle collector for 0.2 mL strips

1 (e.g., Permagen Cat# S500)

Vortex mixer and mini-centrifuge for tubes and strips
Water bath or incubation oven at 65°C
Heat block for microcentrifuge tubes at 60°C✝

Ensure that the thermal cycler and strips allow no more than 4 µL of 30 µL volume evaporation overnight at 65°C

Reagents required
Reagent

Notes

Nuclease-free (“NF”) water

900 µL per reaction

PCR primers to amplify sequencing libraries after capture, e.g.:
Illumina P5: AATGATACGGCGACCACCGA

2.5 uL @ 10 μM per reaction

Illumina P7: CAAGCAGAAGACGGCATACGA

2.5 uL @ 10 μM per reaction

PCR reagents for post-capture amplification

1 per reaction (e.g. Roche Cat# 07958927001)

PCR purification system, e.g., silica columns or SPRI beads

1 cleanup per reaction

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PROCEDURE
PART 1: Hybridization setup
Sequencing libraries are mixed with various blocking nucleic acids, denatured, and then combined with otherhybridization reagents (including baits). These hybridization reactions incubate for several hours to allow
baits to encounter and hybridize with target library molecules.

L1.1 Choose a hybridization temperature (TH)

65°C

62°C

60°C

When bait-target sequence
divergence is expected to beless than 10%

When bait-target sequence
divergence is expected to be
10 to 15%

When bait-target sequence
divergence is expected to be
15 to 25%

L1.2 Prepare materials
Reagents

Equipment

Hyb N and Hyb S from Box 1

Nuclease-free microcentrifuge tubes (×2)

Hyb D and Hyb R from Box 2

0.2 mL strips with attached lids (×1 per 8 reactions)

Block C, Block O, and Block X from Box 2

Pipettors and tips; multichannel for 20 µL recommended

Baits from Box 3 KEEP ON ICE

Vortex mixer and mini-centrifuge for above tube types

Libraries or library pools in 7 µL per reaction

Heat block set to 60°C
Thermal cycler(s); 2 blocks recommended for 24 or more reactions

Program the thermal cycler:
Set lid temperature 5 to 10°C
above each step temperature tominimize evaporation

Step

Parameters

1
2
3
4

60°C, 10 minutes
95°C, 10 minutes
Reduce 0.1°C per second to TH
Hold at TH

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L1.3 Hybridization Reaction Setup
1. Once all reagents have thawed, vortex them to homogenize and then briefly centrifuge.
Heat Hyb N and Hyb S to 60°C and vortex to dissolve any precipitate present after thawing
2. Assemble the Capture Mix in a microcentrifuge (MC) tube combining the reagents in the orderindicated. The following volumes are already adjusted for pipetting error:
MOST TAXAPLANTSSALMONIDS

Component µL / Reaction

Component µL / Reaction

Component µL / Reaction

Hyb N

9.25

Hyb N

9.25

Hyb N

9.25

Hyb D

3.5

Hyb D

3.5

Hyb D

3.5

Hyb S*

0.5

Hyb S*

0.5

Hyb S*

0.5

Hyb R

1.25

Hyb R

1.25

Hyb R

1.25

Block O

2.5

Block O

5.0

Block O-

Block C

2.5

Block C-

Block C

2.5

Block X

0.5

Block X

0.5

Block X

0.5

NF Water-NF Water-NF Water

2.5

Baits

5.5

Baits

5.5

Baits

5.5

TOTAL

25.5

TOTAL

25.5

TOTAL

25.5

*Cloudiness caused by Hyb S addition will clear after step 3

Briefly vortex and centrifuge to collect.
3. For each capture reaction, aliquot 23 µL of Capture Mix to a 0.2 mL well/tube.
4. Add 7 µL of individual or pooled libraries to each Capture Mix aliquot and mix by gently pipetting.
5. Place the reactions in the thermal cycler and run the thermal program, incubating overnight.
Set lid temperature 5 to 10°C above
each step temperature to minimize
evaporation

Step

Parameters

1
2
3
4

60°C, 10 minutes
95°C, 10 minutes
Reduce 0.1°C per second to TH
Hold at TH

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PART 2: Bind and Wash (“Cleanup”)
Bait-target hybrids are bound to streptavidin-coated magnetic beads, and then most non-target DNA isremoved with several rounds of washing with a warm buffer. This is usually performed the day following
completion of Part 1.
For long insert libraries, mix reactions using gentle pipetting or inversion rather than vortexing inorder to minimize shearing effects

L2.1 Choose a wash temperature (TW - typically identical to TH)

65°C

62°C

60°C

When bait-target sequence
divergence is expected to be
10% or less

When bait-target sequence
divergence is expected to be
10 to 15%

When bait-target sequence
divergence is expected to be
15 to 25%

L2.2 Prepare materials
Start at least 90 minutes before intended hybridization stop time
Reagents
Hyb S (Box 1) *Binding Buffer (Box 1) *Wash Buffer (Box 1) *Beads (Box 1)* Allow these reagents to come to room temperature
before use; warm to 60°C and vortex to dissolve
precipitate if necessary

Nuclease-free (NF) Water (up to 900 µL per reaction)

Equipment
Water bath or incubation oven set to the TW (e.g., 65°C)
Receptacles for 50 mL tubes, 0.2 mL strips and microcentrifuge tubes compatible with above incubation device
Vortex mixer and mini-centrifuge for 0.2 mL strips and MC tubes
Magnetic particle collector(s) (MPC) for above strips and/or tubes
When using only amicrocentrifuge (MC) tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 44 cleanups
Nuclease-free MC tubes, 1 per reaction
Heat block set to the TW
Pipettors and tips for 20 – 500 µL

When using a
0.2 mL tube-compatible MPC
Nuclease-free 50 mL tube, 1 per 68 cleanups
Nuclease-free 0.2 mL PCR strips withindividually-attached lids, 1 vessel per reaction
Thermal cycler set to TW
Pipettors and tips for 20 – 200 µL;
multichannel pipettor strongly recommended

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L2.3 Wash Buffer X preparation
This step generates enough Wash Buffer X for 44 reactions in microcentrifuge (“MC”) tube cleanup format,
and 68 reactions in 0.2 mL cleanup format; scale up or down if needed.
1. Thaw and thoroughly homogenize Wash Buffer and Hyb S prior to aliquoting in order to dissolve any
visible precipitate; warm slightly if necessary.
2. Combine 400 µL Hyb S, 39.6 mL NF water and 10 mL Wash Buffer in a 50 mL tube. Vortex
thoroughly, label “Wash Buffer X.”
Wash Buffer X can be stored at 4°C for 1 month
3. Heat the Wash Buffer X to the TW in the water bath or oven for at least 30 minutes before use.

L2.4 Bead preparation

Prepare beads immediately prior to use

1. For each capture reaction, aliquot 30 µL beads to a microcentrifuge tube.
2. Pellet the beads in the MPC until the suspension is clear (1-2 minutes).
Leaving the tubes on the magnet, remove and discard the supernatant.
3. Add 200 µL Binding Buffer to each bead aliquot. Vortex to resuspend the beads and centrifuge
briefly. Place tube in the MPC and pellet beads; remove and discard the supernatant.
4. Repeat Step 3 above twice for a total of three washes.
5. Resuspend each washed bead aliquot in 70 µL Binding Buffer. If proceeding to washing in 0.2 mL
format, transfer the aliquots to PCR strips.
TIP: Beads can be prepared in 8 (or fewer) reaction batches (240 µL) in a microcentrifuge tube. Multiply allvolumes by the number of reactions in the batch; i.e., for 8 reactions-worth, wash with 1.6 mL and resuspend in
560 µL Binding Buffer, then aliquot 70 µL suspension to individual tubes.

L2.5 Binding beads and hybrids
1. Heat the bead aliquots to the TW (e.g., 65°C) for at least 2 minutes.
2. Transfer each capture reaction to the heated bead aliquots. Mix by pipetting.
3. Incubate the libraries + beads on the heat block or thermal cycler for 5 minutes. Agitate at the 2.5
minute mark by flicking or inverting to keep the beads suspended, followed by briefly centrifuging.

L2.6 Bead washing
1. Pellet the beads with the MPC until the solution is clear. Remove and discard the supernatant.
2. Add 375 µL (MC tube format) or 180 µL (0.2 mL format) warmed Wash Buffer X to the beads,
remove from the MPC, place on heat block for 15 seconds, and mix by pipetting. Briefly centrifuge
to collect.
3. Incubate for 5 minutes at the TW in the heat block or thermal cycler. Agitate at the 2.5 minute markby gentle pipetting. Briefly centrifuge to collect the mixture.
4. Repeat steps 1 through 3 two times for MC tube format (three washes total), or three times for 0.2
mL format (four washes total). After the last wash and pelleting, remove as much liquid as
possible without touching the bead pellet.

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PART 3: Library Resuspension and Amplification
Bead-bound enriched library is resuspended in Buffer E and amplified.

L3.1 Prepare materials
Reagents

Equipment

Buffer E (Box 2)

Tubes appropriate for PCR master mix assembly

PCR primers for amplifying libraries (e.g., P5 and P7)

Tubes or strips for 50 µL PCR amplification

PCR reagents for post-capture amplification

Pipettors and tips capable of 5 – 100 µL volumes

PCR purification system, e.g., silica columns or SPRI beads

Vortex mixer and mini-centrifuge for above tube types
Thermal cycler

S3.2 Enriched library recovery
1. Add 30 µL Buffer E to the washed beads and thoroughly resuspend by pipetting.
Then, depending on your library amplification system, choose workflow A or B:
WORKFLOW A: When using KAPA HiFi HotStart or NEB
Ultra II Q5 polymerase systems for amplification

WORKFLOW B: When not using the polymerase
systems for amplification in workflow A

2B.
2A. Proceed directly to section L3.3 using
this bead resuspension as template inamplification

3B.

Incubate the suspension at 95°C
for 5 minutes
Immediately pellet the beads in the MPC
and collect the supernatant containing
the enriched libraries

L3.3 Library amplification
This is an example post-capture amplification using KAPA HiFi HotStart ReadyMix and Illumina libraries:
1. Assemble the following PCR master mix:
Component

Final ConcentrationµL / Reaction

Sequence

NF Water-

5-

2X KAPA HiFi HotStart Ready Mix

1X

25

-

P5 library primer (at 10 μM)

500 nM

2.5

AATGATACGGCGACCACCGA

P7 library primer (at 10 μM)

500 nM

2.5

CAAGCAGAAGACGGCATACGA

-

10 *

-

50

-

Enriched Library (on- or off-bead)

TOTAL*Remaining bead-bound library can be stored at -20°C for several months.

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L3.3 Library amplification (continued)
2. Cycle the reactions with the following thermal program:
Step

Temperature

Time

1

98°C

3 minutes

295°C

30 seconds

3

62°C

20 seconds

4

68°C

10 minutes

6

8°C

∞

×25

cycles**Minimize cycles where possible.
Cycles required to meet molarity
requirements of sequencing
platform may exceed 25.

3. After amplification:- If beads were included in the amplification reaction and you intend to use silica columns
for purification, pellet the beads first and purify only the supernatant.
-

Otherwise, purify the reaction using your preferred PCR cleanup (e.g., silica columns or
SPRI beads).

The enriched libraries are now ready for quantification, quality-assessment, additional size-selection(if required), and then platform-specific library preparation and sequencing.
If insufficient total mass was acquired from a single amplification reaction for e.g. PacBio or
Oxford Nanopore library preparation, perform additional amplifications using the remaining
non-amplified enriched library.

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APPENDIX
LA1: Troubleshooting
During hybridization, my thermal cycler dropped below the hybridization temperature.
You can expect a lower on-target read proportion and target read complexity for these libraries thanif the temperature had remained where intended, but not outright enrichment failure. Shallow preliminary
sequencing will determine whether targets are likely to be retrieved at sufficient coverage within budget.
My enriched and amplified library is not visible on electrophoresis gel or similar.
Successful captures frequently yield a total mass of just a few nanograms even after
re-amplification, which can be difficult to visualize with electrophoresis. This is most common when
capturing especially small targets (<100 bp), or targets that are present at low frequency in the starting
library (like those in degraded/ancient/environmental DNA), or if there is under-reamplification of the librarypost-capture. Often a few more cycles of library amplification will render the captured product sufficiently
high in concentration to view with electrophoresis. Alternatively, determine with library qPCR whether the
library is of sufficient mass for sequencing. If cycling is halted before reaching PCR plateau, the qPCR
product can be visualized with electrophoresis to determine length distribution. Consult with yoursequencing provider for library concentration and volume requirements.
My enriched and amplified library appears significantly longer than my original library, or has two peaks.
This may happen if the libraries are over-amplified and have formed ‘daisy-chains’ or ‘bubbles’ by
experiencing cycles of denature-renature without template extension. These can be reverted to their original
appearance in electrophoresis by applying three PCR cycles using regular library amplification.
I observe a high ratio of PCR duplicates in my enriched library sequence data.
Percent duplicates in sequencing data (i.e. “clonality” or “duplication rate”) increases as you
sequence deeper, and therefore it can only be fairly compared between experiments when the sequencing
depth is normalized before analysis. Evaluate whether you have simply over-sequenced the libraries byplotting raw sequencing reads obtained on the X axis, and unique reads observed on the Y axis. If thiscomplexity curve has plateaued, but you achieved sufficient unique reads, you sequenced more deeply thannecessary. If it has not flattened, or you need to increase the total potential unique read yield of the library,use more DNA per library preparation and/or more library per capture reaction. Avoid diluting baits before
capture. When working with heavily contaminated or damaged DNA target molecules, consider reducing
temperatures used in all steps to improve capture sensitivity. Reducing PCR cycles when possible may also
improve target coverage uniformity and complexity for a given sequencing depth, in some cases having anindirect effect on duplication rate. For more information about library complexity for any NGS application,
we recommend Daley & Smith 2013 (doi: 10.1038/nmeth.2375).

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LA2: myBaits Procedure Quick Guide - Long Insert Protocol
1.

For each reaction, build in the following Capture Mix appropriate to your target taxon; pipetting error is built in:
MOST TAXA
ComponentµL / Reaction
Hyb N
9.25
Hyb D
3.5
Hyb S
0.5
Hyb R
1.25
Block O
2.5
Block C
2.5
Block X
0.5
NF Water
Baits
5.5
TOTAL
25.5

PLANTS
ComponentµL / Reaction
Hyb N
9.25
Hyb D
3.5
Hyb S
0.5
Hyb R
1.25
Block O
5.0
Block C
Block X
0.5
NF Water
Baits
5.5
TOTAL
25.5

SALMONIDS
ComponentµL / Reaction
Hyb N
9.25
Hyb D
3.5
Hyb S
0.5
Hyb R
1.25
Block O
Block C
2.5
Block X
0.5
NF Water
2.5
Baits
5.5
TOTAL
25.5

2.

For each enrichment reaction, aliquot 23 µL of Capture Mix to their own tubes.

3.

To each Capture Mix aliquot, add 7 µL of each library or library pool.

4.

Incubate the reactions in the thermal cycler for 10 minutes @ 60°C, then 10 minutes @ 95°C, and then drop to
the hybridization temperature (e.g., 65°C) at a rate of 0.1°C per second. Be sure to use a heated lid. Incubate at
the hybridization temperature for 16-24 hours.

5.

1.5 hours before step 9, prepare Wash Buffer X by combining 400 µL Hyb S, 39.6 mL nuclease-free molecularbiology-grade water and 10 mL Wash Buffer in a 50 mL tube. Vortex thoroughly and warm to the hybridization
temperature for at least 45 minutes.

6.

Prepare 30 µL of beads per reaction by washing three times in 200 µL Binding Buffer. Resuspend the washedbead aliquots in 70 µL Binding Buffer and warm the suspensions to the hybridization temperature for at least 2
minutes.

7.

Combine the warmed beads with the hybridization reactions and incubate for 5 minutes at the hybridization
temperature, agitating at 2.5 minutes to keep beads suspended.

8.

Pellet the beads and remove the supernatant. If using microcentrifuge tubes for cleanup, wash the beads threetimes with 375 µL warmed Wash Buffer X, incubating 5 minutes at the hybridization temperature. Wash fourtimes with 180 µL washes if using a 96-well magnetic particle concentrator and 0.2 mL strips/tubes.

9.

Resuspend the beads in 30 µL Buffer E and then use 10 µL of this in a 50 µL library amplification reaction with
KAPA® HiFi or NEB Ultra II Q5 polymerase systems. If not using these polymerase systems, instead elute thelibrary from the beads by incubating the suspension for 5 minutes at 95°C, immediately pellet the beads, andthen use 10 µL of the supernatant in a 50 µL amplification reaction.

10. Purify the amplification reactions using silica columns or SPRI beads. If using silica columns and beads were
included in the amplification reaction, pellet the beads first and purify only the supernatant. The enrichedlibraries are now ready for quantification, quality-assessment, and sequencing.

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