Library preparation (dsDNA double indexing, full-UDG, 2x split)

Marcel Keller; Christiana L Scheib

May 08, 2023

Library preparation (dsDNA double indexing, full-UDG, 2x split)

Forked from Library preparation (dsDNA single indexing, full-UDG, no split)

DOI

https://dx.doi.org/10.17504/protocols.io.8epv5jpwdl1b/v1

Library preparation (dsDNA double indexing, full-UDG, 2x split)

Marcel Keller^1,2,
Christiana L Scheib^1,3

¹Institute of Genomics, University of Tartu;
²Institute of Forensic Medicine, University of Bern;
³University of Cambridge

Marcel Keller

Institute of Forensic Medicine, University of Bern, Institut...

DOI: https://dx.doi.org/10.17504/protocols.io.8epv5jpwdl1b/v1

Protocol Citation: Marcel Keller, Christiana L Scheib 2023. Library preparation (dsDNA double indexing, full-UDG, 2x split). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jpwdl1b/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: April 05, 2023

Last Modified: May 08, 2023

Protocol Integer ID: 80053

Keywords: ancient DNA, aDNA, archeogenetics, archaeogenetics, paleogenetics, palaeogenetics, library preparation, dsdna double indexing, dna libraries for illumina, stranded dna library, dsdna, treatment with udg, dna, dna damage, dna damage in form, udg, double indexing, sequencing, protocol for the preparation, library preparation, deaminated cytosine

Abstract

Protocol for the preparation of double indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448). This protocol does include treatment with UDG (USER) to remove DNA damage in form of deaminated cytosines.

Guidelines

Please read the general guidelines for working in the Ancient DNA protocol collection – University of Tartu, Institute of Genomics.

Materials

Reagents:
ABCDEFG
StepReagentsConc.UnitManufacturerKit/full descriptionProduct number
End repair 1WaterFisher chemicalsWater for HPLC 1L10367171
End repair 1NEB Buffer 210XNew England Biolabs
B7002S
End repair 1dNTPs25mMThermo FisherR1121
End repair 1BSA20mg/mlThermo FisherB14
End repair 1ATP10mMNew England Biolabs
P0756S
End repair 1T4 PNK10U/µlNew England Biolabs
M0201L
End repair 1USER enzyme1U/µlNew England Biolabs
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4)E7335S, E7500S, E7710S, E7730S
End repair 2T4 Polymerase10U/µlNew England Biolabs
M0201L
Adapter LigationQuick Ligation Buffer5.0XNew England BiolabsNEBNext Quick Ligation ModuleE6056L
Adapter LigationAdapter Mix (2.5 µM)2.5µM*Custom made from Eurofins adaptersn/a
Adapter LigationQuick T4 Ligase1.0UNew England BiolabsNEBNext Quick Ligation ModuleE6056L
Fill inWaterFisher chemicalsWater for HPLC 1L10367171
Fill inThermopol Buffer 10X10XNew England BiolabsBst DNA Polymerase, Large FragmentM0275
Fill indNTP 25mM25mMThermo FisherR1121
Fill inBst DNA polymerase8U/µlNew England BiolabsBst DNA Polymerase, Large FragmentM0275
PCRWaterFisher chemicalsWater for HPLC 1L10367171
PCR10x PCR buffer10XEurogentecHGS Taq Diamond setTAQ-I011-1000+
PCRMgCl225mMEurogentecHGS Taq Diamond setTAQ-I011-1000+
PCRBSA 20 mg/ml20mg/mlThermo FisherB14
PCRdNTPs (10 mM)40mMThermo FisherR0191
PCRHGS Taq Diamond1UEurogentecHGS Taq Diamond setTAQ-I011-1000+
PCRDual Index Primers Set 110µMNew England BiolabsNEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)E7600S
PCRDual Index Primers Set 210µMNew England BiolabsNEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2)E7780S
PurificationPB BufferQiagenMinElute PCR Purification Kit19066
PurificationPE BufferQiagenMinElute PCR Purification Kit19065
PurificationEB BufferQiagenMinElute PCR Purification Kit28006
Equipment and consumables:
AB
NumberEquipment and consumables
10.2 ml tube rack
21.5 ml tube rack
11.5 ml cool block 
10.2 ml cool block
10 µl filter tips
20 µl filter tips
100 µl filter tips
200 µl filter tips
1000 µl filter tips
[# of samples]×5 (tubes)0.2 µl PCR strips (8 tubes)
[# of samples]×2+51.5 ml tubes
15 ml tube
[# of samples]×2MinElute columns
150 ml Falcon (waste)

Lab equipment:
Laminar flow hood
Centrifuge (2/1.5 ml)
Heat block
Mini table centrifuge/vortexer
Thermocycler
10 µl pipette
20 µl pipette
100 µl pipette
200 µl pipette
1000 µl pipette

Other consumables:
DNA ExitusPlus
Paper towels

Safety warnings

Reagents

NaOCl (bleach) solution (6%)
- H290 May be corrosive to metals.
- H314 Causes severe skin burns and eye damage.
- H411 Toxic to aquatic life with long lasting effects.
- EUH206 Warning! Do not use together with other products. May release dangerous gases (chlorine). Remove from surface after recommended incubation time with water-soaked tissue.
  


DNA ExitusPlus
H319 Causes serious eye irritation.

Guanidinium hydrochloride (GuHCl ) (in PB buffer of Qiagen MinElute kit)
- H302 Harmful if swallowed.
- H332 Harmful if inhaled.
- H315 Causes skin irritation.
- H319 Causes serious eye irritation.

 
Ethanol
- H225 Highly flammable liquid and vapor.
- H319 Causes serious eye irritation.
 


Equipment
UV radiation
- UV radiation can damage eyes and can be carcinogenic in contact with skin. Do not look directly at unshielded UV radiation. Do not expose unprotected skin to UV radiation. 
- UV emitters generate ozone during operation. Use only in ventilated rooms.
 

Before start

Previous step:
This protocol follows the extract purification protocol.

Following step:
This protocol ends with the PCR setup. Proceed with the PCR and library purification protocol.

Preparations:
Get the following consumables and equipment:

AB
NumbersEquipment and consumables
10.2 ml tube rack
21.5 ml tube rack
11.5 ml cool block 
10.2 ml cool block
10 µl filter tips
20 µl filter tips
100 µl filter tips
200 µl filter tips
1000 µl filter tips
[# of samples]×5 (tubes)0.2 µl PCR strips (8 tubes)
[# of samples]×2+51.5 ml tubes
15 ml tube
[# of samples]×2MinElute columns
150 ml Falcon (waste)

Preparation

Turn the hood on full power and open the glass.

Spray hood and table bench surfaces with DNA Exitus, let sit a minute and wipe down with paper towels.

Wipe down outside surfaces of reagents/tips with DNA Exitus and place in the hood. 

Label the following tubes:
5×1.5 ml tubes: ER, AL, FI, EB-1, EB-2
1×5 ml tube: PCR
0.2 ml PCR strips: ER, AL, FI, PCR-A, PCR-B
Label the 50 ml waste tube, PB tube and PE tube.

Aliquot EB buffer in tubes EB-1 and EB-2: each [# of samples]x30 µl plus 10%

Aliquot water for Master Mixes:
ER:  [# of samples]×25.85 µl plus 10%
FI: [# of samples]×12.20 µl plus 10%
PCR:  [# of samples]×84.00 µl plus 10%

Aliquot PB buffer to a 50 ml tube: [# of samples]x1000 µl plus 10%

Prepare PE (wash) buffer by adding ethanol.

Aliquot PE buffer to a 50 ml tube:  [# of samples]x1380 µl plus 10%

Take DNA extracts out of the freezer to thaw at room temperature. Change gloves.

Blunt End Repair 1

Use 1.5 ml ER tube to set up the Blunt End Repair Master Mix On ice   .

ABCDEF
REAGENTSTOCKFINALUNIT1× VOL (µl)NOTE
Water [already added]25.85
NEB Buffer 2101.0X7.50vortex
dNTPs250.30mM0.90vortex
BSA200.20mg/ml0.75vortex
ATP101.00mM7.50vortex
T4 PNK100.40U/µl3.00on ice
USER enzyme10.06U/µl4.50on ice
Master Mix total50.00
Template DNA or water25.00
REACTION TOTAL75.00
Calculate +10% for all Master Mix components.

Add 20 µl Master Mix to each tube of the ER strip.

Vortex and spin down DNA extracts, add 25 µl of template DNA or water to each tube.

Mix carefully by resuspending, remove bubbles and spin down.

Incubate at 37 °C   for 03:00:00  .

Note
Use free time for preparing Master Mixes for the following steps. Master Mixes should be stored in the fridge.

Blunt End Repair 2

Add 3 µl T4 pol to each reaction.

ABCDEF
REAGENTSTOCKFINALUNIT1× VOL (µl)NOTE
T4 DNA Polymerase30.038U/µl3.00on ice
REACTION TOTAL78.00

Incubate at 25 °C   for 00:30:00  , then at 10 °C   for 00:05:00   

Note
Use free time for preparing Master Mixes for the following steps. Master Mixes should be stored in the fridge.

35m

MinElute Purification 1

Take MinElute columns out of the fridge.

Turn on heat block 37 °C    for elution.

Label columns and tubes with sample ID numbers.

Add 500 µl PB buffer (binding buffer) to MinElute column. You can use the same tip.

Add end-repair reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.

Spin at 13000 rpm, 00:01:00 , discard supernatant, change gloves.

Add 690 µl PE buffer (wash buffer), change tip for every sample.

Spin at 13000 rpm, 00:01:00 , discard supernatant, change gloves.

Spin at 13000 rpm, 00:01:00  (dry spin).

Put column in new tube, change gloves.

Elute in 30 µl EB buffer (elution buffer), change tip for every sample.

 Incubate at 37 °C   for 00:10:00   .

10m

Spin at 13000 rpm, 00:02:00  .

Discard the silica column and close the lid.

Note
Potential stopping point, place end-repaired DNA in freezer until ready for next step.

Adapter ligation

Use 1.5 ml AL tube to set up the Adapter Ligation Master Mix On ice  .
 
ABCDEF
REAGENTSTOCKFINALUNIT1× VOL (µl)NOTE
Quick Ligation Buffer5.01.00×10vortex
Adapter Mix 2.50.25µM5vortex
End Repair Enzyme Mix1.00.10U5on ice
Master Mix total20.00
Template DNA or water30.00
REACTION TOTAL50.00
Calculate +20% for all Master Mix components.

Add 20 µl of Master Mix to each tube of the AL strip.

Add 30 µl of end-repaired template DNA or water to each tube.

Incubate at 20 °C   for 00:15:00  .

15m

MinElute purification 2

Take MinElute columns out of the fridge.

Turn on heat block 37 °C    for elution if not turned on already.

Label columns and tubes with sample ID numbers.

Add 500 µl PB buffer (binding buffer) to MinElute columns. You can use the same tip.

Add adapter ligation reaction mix to the PB buffer inside the MinElute columns and mix by resuspending.

Spin at 13000 rpm, 00:01:00 , discard supernatant, change gloves.

Add 690 µl PE buffer (wash buffer), change tip for every sample.

Spin at 13000 rpm, 00:01:00 , discard supernatant, change gloves.

Spin at 13000 rpm, 00:01:00  (dry spin).

Put column in new tube, change gloves.

Elute in 30 µl EB buffer (elution buffer), change tip for every sample.

Incubate at 37 °C   for 00:10:00  .

10m

Spin at 13000 rpm, 00:02:00 .

Discard the silica column, close the lid.

Note
Potential stopping point, place adapter-ligated DNA in freezer until ready for next step.

Fill-in reaction

Use 1.5 ml FI tube to set up the Adapter Ligation Master Mix On ice  .

ABCDEF
REAGENTSTOCKFINALUNIT1× VOL (µl)NOTE
Water [already added]12.2
Thermopol Buffer101.00×5.0vortex
dNTPs250.40mM0.8vortex
Bst polymerase80.32U/µl2.0on ice
Master Mix total20
Template DNA or water30
REACTION TOTAL50
Calculate +20% for all Master Mix components.

 

Add 20 µl Master Mix to each tube of the FI strip.

Add 30 µl of adapter-ligated template DNA or water to each tube.

Incubate at 37 °C   for 00:30:00   , then at 80 °C   for 00:20:00  .

Note
Use free time for preparing the Master Mix for the following step, Master Mixes should be stored in the fridge

50m

Library amplification (PCR)

Use 5 ml PCR tube to set up PCR Master Mix On ice  .

Note
The PCR reaction will be set up with 2x volume and split into two tubes after adding the fill-in reaction mix. Add indices after aliquoting the Master Mix to the PCR-A strip tubes.



ABCDEF
REAGENTSSTOCKFINALUNIT1× VOL (µl)NOTE
Water [already added]17
10x PCR buffer101.00X10vortex
MgCl2252.50mM10vortex
BSA201.00mg/ml5vortex
dNTPs 10 mM400.80mM2vortex
HGS Taq Diamond10.02U2on ice
Universal primer 1.0100.2µM2vortex
Master Mix total48
Indexing primer100.2µM2vortex
Template DNA or water50
REACTION TOTAL100
Calculate +10% for all Master Mix components.

Aliquot 142 µl of Master Mix to the tubes of the PCR-A strip.

Vortex indexes slightly and spin them down with a table centrifuge.

Add 4 µl of i7 indexing primer (10 µM) to the respective tube with Master Mix.

Add 4 µl of i5 indexing primer (10 µM) to the respective tube with Master Mix.

Add 50 µl of adapter fill-in reaction mix or water to the respective tube with Master Mix.

Mix by resuspending and pipet 100 µl of the reaction to the PCR-B strip tubes.

Check that the lids are tightly sealed and take the strips to the modern lab for PCR.

A	B	C	D	E	F	G
Step	Reagents	Conc.	Unit	Manufacturer	Kit/full description	Product number
End repair 1	Water			Fisher chemicals	Water for HPLC 1L	10367171
End repair 1	NEB Buffer 2	10	X	New England Biolabs		B7002S
End repair 1	dNTPs	25	mM	Thermo Fisher		R1121
End repair 1	BSA	20	mg/ml	Thermo Fisher		B14
End repair 1	ATP	10	mM	New England Biolabs		P0756S
End repair 1	T4 PNK	10	U/µl	New England Biolabs		M0201L
End repair 1	USER enzyme	1	U/µl	New England Biolabs	NEBNext Multiplex Oligos for Illumina (Index Primers Set 1-4)	E7335S, E7500S, E7710S, E7730S
End repair 2	T4 Polymerase	10	U/µl	New England Biolabs		M0201L
Adapter Ligation	Quick Ligation Buffer	5.0	X	New England Biolabs	NEBNext Quick Ligation Module	E6056L
Adapter Ligation	Adapter Mix (2.5 µM)	2.5	µM		*Custom made from Eurofins adapters	n/a
Adapter Ligation	Quick T4 Ligase	1.0	U	New England Biolabs	NEBNext Quick Ligation Module	E6056L
Fill in	Water			Fisher chemicals	Water for HPLC 1L	10367171
Fill in	Thermopol Buffer 10X	10	X	New England Biolabs	Bst DNA Polymerase, Large Fragment	M0275
Fill in	dNTP 25mM	25	mM	Thermo Fisher		R1121
Fill in	Bst DNA polymerase	8	U/µl	New England Biolabs	Bst DNA Polymerase, Large Fragment	M0275
PCR	Water			Fisher chemicals	Water for HPLC 1L	10367171
PCR	10x PCR buffer	10	X	Eurogentec	HGS Taq Diamond set	TAQ-I011-1000+
PCR	MgCl2	25	mM	Eurogentec	HGS Taq Diamond set	TAQ-I011-1000+
PCR	BSA 20 mg/ml	20	mg/ml	Thermo Fisher		B14
PCR	dNTPs (10 mM)	40	mM	Thermo Fisher		R0191
PCR	HGS Taq Diamond	1	U	Eurogentec	HGS Taq Diamond set	TAQ-I011-1000+
PCR	Dual Index Primers Set 1	10	µM	New England Biolabs	NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)	E7600S
PCR	Dual Index Primers Set 2	10	µM	New England Biolabs	NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2)	E7780S
Purification	PB Buffer			Qiagen	MinElute PCR Purification Kit	19066
Purification	PE Buffer			Qiagen	MinElute PCR Purification Kit	19065
Purification	EB Buffer			Qiagen	MinElute PCR Purification Kit	28006

	A	B
	Number	Equipment and consumables
	1	0.2 ml tube rack
	2	1.5 ml tube rack
	1	1.5 ml cool block
	1	0.2 ml cool block
		10 µl filter tips
		20 µl filter tips
		100 µl filter tips
		200 µl filter tips
		1000 µl filter tips
	[# of samples]×5 (tubes)	0.2 µl PCR strips (8 tubes)
	[# of samples]×2+5	1.5 ml tubes
	1	5 ml tube
	[# of samples]×2	MinElute columns
	1	50 ml Falcon (waste)

	A	B
	Numbers	Equipment and consumables
	1	0.2 ml tube rack
	2	1.5 ml tube rack
	1	1.5 ml cool block
	1	0.2 ml cool block
		10 µl filter tips
		20 µl filter tips
		100 µl filter tips
		200 µl filter tips
		1000 µl filter tips
	[# of samples]×5 (tubes)	0.2 µl PCR strips (8 tubes)
	[# of samples]×2+5	1.5 ml tubes
	1	5 ml tube
	[# of samples]×2	MinElute columns
	1	50 ml Falcon (waste)

A	B	C	D	E	F
REAGENT	STOCK	FINAL	UNIT	1× VOL (µl)	NOTE
Water [already added]				25.85
NEB Buffer 2	10	1.0	X	7.50	vortex
dNTPs	25	0.30	mM	0.90	vortex
BSA	20	0.20	mg/ml	0.75	vortex
ATP	10	1.00	mM	7.50	vortex
T4 PNK	10	0.40	U/µl	3.00	on ice
USER enzyme	1	0.06	U/µl	4.50	on ice
Master Mix total				50.00
Template DNA or water				25.00
REACTION TOTAL				75.00