TITLE: Illunina library preparation and dual hybridization protocol of ERC GLOBAL 
AUTHORS: Vincent R C Soulé, Cedric Mariac, Thomas LP Couvreur

[DESCRIPTION]
Step by step protocol for library preparation of Annonaceae species within the ERC GLOBAL project. This protocol was followed for all libraries prepared within the project. Each step involves a purification phase  (AMPure XP protocol), and is detailed at the start of this protocol. The protocol is done per series of 48 samples at a time with indexing.

The protocol also describes the steps for the dual hybridization of Daicel Arbor Biosciences Mybaits Angiosperm353 kit + Custom Annonacea baiting kit. The protocol is based on hybridisation protocols used at DIADE research unit IRD Montpellier France and on the Hybridization Capture for Targeted NGS User Manual version 5 protocol "standard" of Daicel Arbor Biosciences Mybaits. 

To proceed DNA extraction of plant and in particular from herbarium and silicagel specimens of annonacea read the Annonaceae DNA extraction protocol from silicagel dried and herbarium preserved leaves 
       Vincent Soulé1,
Thomas LP Couvreur1,
Cedric Mariac1
1DIADE, Univ Montpellier, CIRAD, IRD, Montpellier, France
dx.doi.org/10.17504/protocols.io.5qpvorqx9v4o/v1

[BEFORE_START]
Clean bench and pipettes with water and then ethanol. Follow the instructions for use of the Arbor Biosciences V5 kit.
check if you have all the material needed for all the step or equivalent tools

[GUIDELINES]
This librayry preparaion & dual hybridisation protocol follows and is inspired by Henriks et al (2021) using both the Annonaceae specific baits and the Angiosperms353 baits (see references). In the end we sequence both the Annonaceae specific baits and the Angiosperms353 universal ones.

Both the Annonaceae (Couvreur et al. 2019) and Angiosperms353 (Johnson et al., 2019) bait kits are available as Arbor Biosciences “myBaits” kits (Daicel Arbor Biosciences, Ann Arbor, Michigan, USA; https://arborbiosci.com/genomics/targeted-sequencing/mybaits/).

[STEPS]
SECTION: Part 1: library preparation ,  DNA preparation and shearing
3. Depending on concentration of each sample, dilute the DNA in 0.2 mL microtubes Diagenode suitable for shearing with ultrasound. Target amount of DNA should, if possible, be 3-4 µg in 50 µL (to be supplemented with water). The number of shearing cycles and their duration can be modified depending on the initial degradation state of the DNA and/or the desired fragment size.
Shearing is done with the below equipment:
 

SECTION: Fast End Repair &amp; phosphorylation
4. Prepare a buffer and enzyme mix according to the number of samples to be processed


     For   1 sample:        Sheared   DNA      40μL        End   Repair Reaction Buffer 10X      5μL        End   Repair Enzyme Mix      5μL        Final   Volume =      50μL   

NEBNext End Repair Module  E6050L or E6050S

SECTION: Ligation
5. Depending on the previous dosage, calculate the volume to be taken per sample to ideally obtain 200 ng of DNA in 13.8 µL.
If necessary, dilute samples that are too concentrated .
Careful not to vortex the ligase. Mix by flicking the tube.
SECTION: PCR1 amplification + Purification AMPure
7. In a LightCycler plate® 480 Multiwell plate 96 white arranged on refrigerated stand: deposit 30 µL of each fluorescent standards ref KK2701 (1 to 4) in duplicates in 8 separate wells. Keep the plate away from light.
SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.1. Pulse centrifuge the tubes.
SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.2. Deposit 50 µL of DNA and water if needed to dilute to match concentration to the tubes in the Pico Bioruptor (Diagenode).

SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.3. Start N cycles 15 s ON / 30 s OFF at 4 °C. 
Depending on the DNA extraction control gel 0 < N < 10. 


 


SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.4. Check size using 3 µL DNA + 10 µL DNA dillution buffer AL420 protocol with QIAXCEL, if necessary undertake additional cycles.

Pattern must be between 150 and 600 bp, but depending on the initial degradation, it may be centred on 100 bp.
QIAxcel  QIAGEN (DNA Screening Kit)


SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.5. Restart N+B cycles 15 sON / 30 s OFF at 4 °C.

SECTION: Part 1: library preparation ,  DNA preparation and shearing
3.6. Mesure DNA size again using QIAGEN QIAXCEL Bioanalyser
 3 µL DNA + 10 µL DNA dillution buffer AL420 protocol analysis. 

or other bioanalyser , you can also make a DNA gel



 

SECTION: Fast End Repair &amp; phosphorylation
4.2. Add 40 µL of DNA from previous step, vortex and Spin down the sample (using pcr plate spinner).
SECTION: Fast End Repair &amp; phosphorylation
4.3. Leave 30 min at Room temperature.
SECTION: Fast End Repair &amp; phosphorylation
4.4. Stop the reaction using a purification step AMPure XP  1X (50µL) or 1.8X (90µL) if you want to retain small DNA fragments. See  introduction for details of the purification step using   
SECTION: Fast End Repair &amp; phosphorylation
4.5. Elute the samples in 30 µL water and quantify using Nanoquant.

 



7.9. 

 

 
SECTION: PCR1 amplification + Purification AMPure
7.1. Prepare a volume of Mix according to the number of samples:

     For   1 sample:        DNA      12μL/ 20μL          PE   solution (34) 10μM*      1.5μL/ 2.5μL          INDEX   or PE Solution (35) 10μM*      1.5μL/ 2.5μL          KAPA   HiFi Master Mix 2X      15μL/ 25μL          Final   Volume =      30μL/ 50μL     
Roche KAPA Hifi PCR KIT 250rx
Index and adapter sequence on aftached file
Index used: AD001; AD002; AD003; AD004; AD005; AD006; AD007; AD008; AD009; AD0010; AD0011; AD0012.
SECTION: Introduction and AMPure XP protocol
2. AMPURE XP DNA Purification protocol 
On each steps of the protocol a purification step is need to change solution, concentrate or purify dna. 


Each purification step is done with    

The only changes between the different purification steps is the start volumes as such: volume ratio 1X to 1.8X AMPure. For example for 1.8X: for 100 µl to be purified added 180 µl Ampure


Below we provide a protocol for 1X  
for start volume of 50 µL of DNA
resuspension (elution) volume = 20 µL. 
The protocol is described for 96 well pcr plates, but can also be done in 1.5/ 2mL tubes.

SECTION: Introduction and AMPure XP protocol
2.1. Use    at Room temperature, take out 30 minutes in advance.
Prepare a 70% ethanol solution.

Add 50 µLAMPure XP in each well.

SECTION: Introduction and AMPure XP protocol
2.2. Mix by pipetting or shake and wait 10 min min. quickly spin down 10sec using pcr plate spinner if pcr plate or mini centrifuge for tubes 
DNA will bind to the beads
SECTION: Introduction and AMPure XP protocol
2.3. Place the plate on the magnetic stand and wait 5 min min.
Remove liquid phase by pipetting , avoid pipetting the beads.
SECTION: Introduction and AMPure XP protocol
2.4. Keep on magnetic stand add 150 µL    .
Remove liquid phase by pipetting, avoid pipetting the beads. 

SECTION: Introduction and AMPure XP protocol
2.5. Repeat wash step on magnetic stand and add 150 µL    
Remove liquid phase by pipetting, avoid pipetting the beads. 
Allow to dry for 5 minutes at Room temperature

SECTION: Introduction and AMPure XP protocol
2.6. Take PCR plate out of the magnetic stand and add the needed "elution volume"; here 20 µL of ultra pure water  
Mix by pipetting.

Wait 10 min minutes at Room temperature for beads to resuspend in water. 

SECTION: Introduction and AMPure XP protocol
2.7. The AMPure purification step is over. 
Make sure to place the plate on the magnetic stand to avoid pipetting beads for next usage or quantification. 
Mind that purification step reduces DNA quantity by 20 to 50% and removes small DNA fragments.
To keep small fragments use 1.8X AMPure ratio instead of 1X.
SECTION: Fast End Repair &amp; phosphorylation
4.1. Distribute 10 µL of enzyme and buffer mix in each well.
SECTION: Ligation
5.1. Take the samples, place them on a 96-well plate on a refrigerated stand, and adjust all the volumes to 13 µL with water.
SECTION: Ligation
5.2. Then add to each sample 2 µL of TAG-P5 adapt (4 µM).
SECTION: Ligation
5.3. Prepare a volume of Mix according to the number of samples:
  
     For   1 sample (DNA = 200ng, 13μL):        DNA      13μL   / 13.8μL        PE-P5   adapt 4μM      2μL        MPE7 adapt 40μM      0.2μL        T4   DNA ligase 5U/μL      2μL        Buffer   T4 10X      2μL        H2O      0.8μL   / 0μL        Final   Volume =      20μL   
Update the thawing/freezing number of P7 P5 adapters on their storage box.

NB; Tag referes as P5 6pb barcoded adapter used as index but not read by ilumina ; demultipexed after sequencing.

adapter p7  &  p5 sequences on atached file 

 

 

Thermo Scientific‱ ADN ligase T4 (5 U/µl) EL0013 
SECTION: Ligation
5.4. Deposit 4.2 µL of Mix by well, vortex briefly and pulse centrifuge.
SECTION: Ligation
5.6. Purify 20µL post ligation using    with XP1.8X ratio 
(36 µL of ampure ); 

Then elute  in 25 µLultra pure water.


 

SECTION: Ligation
5.5. Place the plate on the thermocycler and launch the "LIG" program:

     Step      Temperature      Duration        1      +22°C      2H30        2      +65°C      10min        3      +4°C      20min   

SECTION: PCR1 amplification + Purification AMPure
7.2. Deposit 18 µL of Mix per well and pulse centrifuge.
SECTION: PCR1 amplification + Purification AMPure
7.3. Deposit 12 µL of DNA per well, mix with the pipette and pulse centrifuge.
SECTION: PCR1 amplification + Purification AMPure
7.4. Place the plate in the lightcycler and launch the following the "RTLib1" program. 
 
     Step      Temperature      acquisition      Duration      spleen      Cycles        Pre-incubation      98°C      None      45sec      4.4°C/sec      1cycle           amplification      98°C      None      15sec      4.4°C/sec         55   cycles        62°C      None      30sec      2.2°C/s        72°C      Single      30sec      4.4°C/sec   
PCR should be stopped when signal is between standards 1 and 3 (optimal library amplification range), generally between cycles 10-14. more for low quality or low quantity DNA

Important : Do not over amplify, it will reduce you sequencing quality, stop before reachin plateau phase of amplification 


  



SECTION: PCR1 amplification + Purification AMPure
7.5. Stop the run just after the last acquisition (green dot on the graph) when the double-stranded DNA is full length. If necessary, transfer the "passed" samples to another AB 1400 plate by piercing the film with a scalpel blade at each well concerned.
SECTION: PCR1 amplification + Purification AMPure
7.6. Repeat if necessary for samples requiring more subsequent cycles, using the same program but without pre-incubation.
SECTION: PCR1 amplification + Purification AMPure
7.7. Purify using    XP 1X (30 µL); see section 2.
 Eluate the samples in 30 µLof ultra pure water.
SECTION: PCR1 amplification + Purification AMPure
7.8. Perform an analysis on Qiaxcel (3 µL) and dose on Nanoquant (2 µL).

SECTION: Nick Fill-in (Bst DNA Polymerase)
6. Position the 96-well plate on a refrigerated stand.
Prepare a volume of Mix according to the number of samples:

     For   1 sample:        DNA      23.5μL        DNTPs   5mM      1.5μL        Bst   DNA POLYMERASE 8U/μL      2μL        Thermo   Pol Buffer 10X      3μL        Final   Volume =      30μL   
Bst DNA Polymerase, Large Fragment
(NEB#: M0275)
DNTP Mix , REF U151B 10mM U1515 or 1511 (200 or 1000µL)
SECTION: Nick Fill-in (Bst DNA Polymerase)
6.1. Deposit 6.5 µL of Mix per well in a AB1400 pcr plate and pulse centrifuge.
SECTION: Nick Fill-in (Bst DNA Polymerase)
6.2. Deposit 23.5 µL of DNA per well, mix with the pipette and pulse centrifuge.
SECTION: Nick Fill-in (Bst DNA Polymerase)
6.3. Place the plate on the thermocycler and launch the "BST" program:
 
 
     Step      Temperature      Duration        1      +37°C      15min        2   /optional/      +10°C      20min   

SECTION: Nick Fill-in (Bst DNA Polymerase)
6.4. Purify using    XP 1X (30 µL); see section 1.
Elute in 15 µLultra pure water.


 



 
SECTION: Preparation of Bulk
8. Prepare an equimolar mix of all 48 sample by adding 1 µLto 25 µLof each purified sample from PCR1 step. Volume will depend of concentration and size of DNA. 
SECTION: Preparation of Bulk
8.1. Match 0.5 pmol per samples  ex : 133 ng for 400pb DNA.

Each volume is added in a 1.5 mL DNA low bind tube, total volume between 100 µL to 1000 µL
 
SECTION: Preparation of Bulk
8.3. 
 

SECTION: Part 2: Hybridization step for targeted DNA sequencing
9. This part of the protocol describes dual hybridization process for targeted DNA sequencing using Daicel Arbor Biosciences Mybait KIT V5 Angiosperm353 and custom Annonacea kit.
In this protocol we use a bulk of 48 samples for each reaction (Arbor recommend max 8 sample per reactions). This protocol describes the quantity for one reaction or 8 reactions (48 or 384 samples) 

A total of5.5 µLof baits is required per reaction. We used a ratio of a 3/4 Angiosperms353 baits and 1/4 Annonaceae baits per reaction following Henriks et al. (2021):

4.125 µL of Angiosperms353 baits
1.375 µL of Annonaceae baits
SECTION: Bait mix preparation
10. Program the Thermo Mixer so that it reaches 65 °C and a second Thermo mixer to 95 °C. Place the Arbor Biosciences Baits on ice.
Heat the Hyb N and Hyb S bait reactives at 60 °C and vortex to dissolve the aggregates.
Prepare a volume of Mix "HYB" according to the number of reactions planned, vortex and centrifuge:
 
 
     Component      µL / Reaction               Hyb N      9.25        Hyb D      3.5        Hyb S*      0.5               Hyb   R      1.25               Baits      5.5               TOTAL      20          

SECTION: Bait mix preparation
11. Incubate the HYB mix 10 min at 60 °C and vortex to recover the condensate
For each capture reaction, alicot  18.5 µL in 1.5 µL of DNA in a low bind tube.

We refer to these reaction aliquots of hybridization Mix as "HYBs"

Let stand 5 min at Room temperature 
Prepare a volume of Mix "Block" according to the number of reactions of capture, vortex and centrifuge:
 
     BLOCK   // For 1 capture:        Block O      5        Block X      0.5        INDEX 100μM      0.5        V   total      6   
For each capture reaction, aliquot 5.0 µL of "Block" in a 0.5mL tube.
SECTION: Hybridization
12. Add 7 µL of BULK DNA library 50-100 ng/µL to each 5 µL aliquot of "Block" and homogenize by pipetting. We then obtain a Mix of 12 µL

These libraries with Blocker Mix aliquots are now called "LIB" (block + libraries)

Place the "LIB" on the second Thermo Mixer at 95 °C for 5 min.
Transfer the "LIB" and the "HYBs" mix into the Thermo Mixer at 65 °C 5 min   

    
SECTION: Capture and washing
13. Bring Hyb S (the reactive, not the mix), Buffer E and wash buffer to Room temperature in order to prepare the washing solutions about 1h30 before stopping the hybridization reaction.

Prepare the Wash Buffer X in a falcon tube of 15 mL
 
     For   3 captures:        HYB S      40μL        Water      3.96mL        Wash   Buffer      1mL        Final   Volume =      5mL   

Wash Buffer X can be stored at 4 °C for up to 1 month
SECTION: Elution
14. Add 25 µL of Buffer E box -20° (bring back to Room temperature before hand)
Incubate at 98 °C for5 min on ThermoMixer or Heating Block (use lid).
Place the tubes on a magnetic stand and quickly recover the supernatant containing the enriched libraries and transfer it into a new PCR tube / plate.
SECTION: Capture and washing
13.1. Vortex and place the Wash Buffer X at hybridization temperature (+65 °C in a water bath) at least 45 min before use.
Vortex to homogenise capture beads (kit , box at 4°C).

For each capture reaction, aliquot 30 µL of beads (Box at 4 °C) 

TIP: Beads can be prepared in 8 (or fewer) reaction batches (240 µL) in a 1.5 µL tube. Multiply all volumes by the number of reactions in the batch; i.e., for 8 reactions-worth, wash with 1.6 mL and resuspend in 560 µL μL Binding Buffer, then aliquot 70 μL suspension to individual tubes.
SECTION: Capture and washing
13.2. Arrange tubes on a magnetic stand for 1 min to 2 min until the aggregate is formed and remove the supernatant.
Repeat 3 consecutive times:
Add 200 µL of Binding Buffer in each tube, vortex 3 s, centrifuge briefly and finally eliminate the supernatant on magnetic stand.
Resuspend beads in 70 µL of Binding Buffer.
SECTION: Capture and washing
13.4. Repeat 3 consecutive times:
Add 375 µL of Wash Buffer X (heated) in each tube, vortex, centrifuge briefly, incubate at hybridization temperature on Thermo Mixer for 5 min minutes at 1200 rpm and finally eliminate the supernatant on magnetic stand.
SECTION: Capture and washing
13.3. Place the beads at hybridization temperature on Thermo Mixer at least 2 min before transfer.
Quickly transfer each capture reaction (30 µL) into a tube containing the beads and homogenize by pipetting.
Incubate the library and beads mixture at hybridization temperature of 65 °C on Thermo Mixer for 5 min at 1200 rpm (program "CAPT").

Eliminate the supernatant on magnetic stand.
SECTION: RT-PCR 2 (final)
15. In a LightCycler plate® 480 Multiwell plate with 96 arranged on refrigerated plate.

Deposit 50 µL of each fluorescent marker 1 to 4 repeated twice in 8 separate wells on the plate. Keep the plate away from the light.
Prepare a Mix for each INDEX used. There are 12 Illunima INDEX that can be used.
 
     By   well:        BULK   DNA      20μL         PE   solution (34) 10μM      2.5μL         INDEX   10μM or PE Solution (35) 10μM*      2.5μL         KAPA   HiFi Master Mix 2X      25μL         Final   Volume =      50μL    
index and adapter sequence on atached file 
Index used: AD001; AD002; AD003; AD004 AD005 AD006 AD007 AD008 AD009 AD0010 AD0011 AD0012 
Deposit 30 µL of Mix (index+PE34+kapa pcr mix) per well and pulse centrifuge.

Deposit 20 µL of DNA per well, mix with the pipette and pulse centrifuge.

Place the plate at the lightcycler and launch the following  program:

     Step      Temperature      acquisition      Duration      spleen      Cycles        Pre-incubation      98°C      None      45sec      4.4°C/sec      1cycle           amplification      98°C      None      15sec      4.4°C/sec         55   cycles        62°C      None      30sec      2.2°C/s        72°C      Single      30sec      4.4°C/sec   

 Stop the run before the plateau phase. Generally 10 to 16 cycles. If necessary, add some extra cycles for some samples. Important : Do not over amplify, it will reduce you sequencing quality.

 
Carry out a purification step using   XP 1X on half the volume 25μL , keep the leftover volume (approx. 25 µL) at -20 °C as backup.
SECTION: Preparation of bulk for illunima sequencing
16. Prepare an equimolar mix of all samples.
We end up with a single tube with all indexes mixed, and ready to sequence.

Refer to your sequencing platform for final volume and concentration.
In our case we needed a minimum of 50 µL and DNA fragments should be at 400pb. 
All samples were sequenced with Novogen using a NovaSeq PE150.

SECTION: Hybridization
12.1. Transfer 18.5 µL of each "HYBs" to each corresponding "LIB" and homogenize by pipetting 5 times.
Incubate at hybridization temperature of 65 °C  5 min (16h-24h).  

 

SECTION: RT-PCR 2 (final)
15.1. Proceed with the purification with 1.5 mL DNA low bind tube as follows:

Prepare the sample: Mix the 25µL DNA sample with 25µL AMPure.
Bind: Incubate the mixture for 10 minutes at room temperature to allow the DNA to bind to the beads and put on magnetic stand;
Wash: after 5 minutes on magnetic stand remove supernatant;
Wash the beads twice with 70% ethanol to remove impurities;
Let dry for 5 minutes to remove residual ethanol and dry the purified DNA;
Remove from magnetic stand and resuspend the dried DNA by adding 20 µL water. 
 
 Let DNA elute at least 5 min min.
 
 Perform a quantification with Nanodrop and quality analysis on Qiaxcel.

SECTION: Preparation of Bulk
8.2. Purify using    XP 1.8X; see section 2. Volumes depends on the total volume of the bulk.
 Eluate the samples in 30 µLof ultra pure water.

 
   
SECTION: Introduction and AMPure XP protocol
1. Library preparation protocol is carried out after the DNA extraction detailed in : 
 
To start we need purified DNA dilluted in water or TE buffer 50µL or more at 2 to 900ng/µL 
below 60ng/µL samples are considered low quantity if only diluted in 50µL
samples with size less than 200pb is considered low quality/small fragments dna (use 1.8X ampure)