Sep 09, 2025

Public workspaceIndexing of ancient DNA libraries

DOI
https://dx.doi.org/10.17504/protocols.io.q26g79qekvwz/v1
  • Michael V. Westbury1,
  • Alba Rey-Iglesia1,
  • Deon de Jager1,
  • Vanssy Li1,
  • Eline Lorenzen1
  • 1University of Copenhagen
  • Lorenzen Lab
Deon de Jager
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g79qekvwz/v1
Protocol Citation: Michael V. Westbury, Alba Rey-Iglesia, Deon de Jager, Vanssy Li, Eline Lorenzen 2025. Indexing of ancient DNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79qekvwz/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: June 26, 2025
Last Modified: September 09, 2025
Protocol Integer ID: 221107
Keywords: ancient DNA, Indexing libraries, indexing of ancient dna library, indexed ancient dna library, index ancient dna, ancient dna library, p7 index primer, sequencing library, ancient dna, libraries with the p5, dna, indexing, pcr mixture, pcr, index, p5
Abstract
This protocol describes how to index ancient DNA sequencing libraries with the P5 and P7 index primers mix. All the reagents and PCR mixture should be prepared in the clean lab, and then the PCR must be run in the modern lab. The output of this protocol is indexed ancient DNA libraries.
Guidelines
If it is an ancient sample, prepare the PCR mixture in the clean lab and run in the modern lab.
Protocol materials
ReagentNEBNext Q5U Master Mix – 250 rxnsNew England BiolabsCatalog #M0597L
ReagentKAPA HiFi HotStart Uracil ReadyMix (2X)RocheCatalog #07959079001
Troubleshooting
Before start
  • Make sure you have the P5+P7 index primers mix (10 uM) ready before you start.
  • If you are going to sequence in the following steps, avoid using the same index for different libraries in one sequencing pool.
Prepare the PCR master mix

Note
Prepare the reagents and mixture in the clean lab and run the PCR in the modern lab.
Index PCR master mix setup:
  • N samples = No. of libraries (incl. extraction and library build negative controls) + index PCR negative control (no library template - use your ddH2O as the "template").
  • Required reagents:
  1. ReagentNEBNext Q5U Master Mix – 250 rxnsNew England BiolabsCatalog #M0597L or ReagentKAPA HiFi HotStart Uracil ReadyMix (2X)RocheCatalog #07959079001 or any uracil-tolerant polymerase.
  2. ddH2O.
  3. P5 and P7 indexing primers mix (10 μM).

ReagentStock Conc.Volume (μL)N sample
ddH2O88 * (n*1.1)
Q5U Master Mix2525 * (n*1.1)
Total volume3333 * (n*1.1)
Aliquot 33 uL of the master mix into PCR tubes, then only add the unique primer mix for each tube.
P5+P7 index primer mix10 μM2
DNA template15
Total volme50

Add 33 μL of indexing PCR master mix to a PCR tube (as stated in the table above).
Add 2 μL of the P5+P7 index primer mix to each PCR tube (as stated in the table above). Make sure that each sample gets a unique index combination.
Add 15 μL of the prepared DNA (library) template to the PCR tube. For the indexing negative control, use ddH2O. The final volume of each PCR tube is 50 μL.
Put the PCR tubes in a ziploc bag and label it with your name, date, sample IDs, etc. to take it to the modern lab.
Running PCR
Run the PCR in the modern lab.

For Q5U:

StepTempTimeCycles
A*98 °C60 s* Pre-Heat Step
B98 °C45 s
C98 °C15 s** xCycles (12-18)
60 °C30 s
72 °C20 s

* Pre-Heat Step: Step A is for Q5U specifically, as the enzyme’s documentation says that the PCR machine should be pre-heated to 98°C before the samples go in, so the samples should be placed into the PCR machine quickly just before the end of this step, which is then followed by the 45s 98°C step.

** The number of cycles depends on your libraries. (If you want to accurately determine the number of cycles, you can do a qPCR first.)
12-18 cycles are sufficient for ancient DNA amplification. A higher number of cycles will lead to higher duplication rates in your sequencing data. If the libraries will be neriched for mitochondrial DNA, this is not usually a problem, as you will have a high duplication in the post-capture libraries, and you want a high concentration to do the capture/enrichment, so many indexing cycles are a good idea. If you are not capturing and instead doing shotgun for nuclear deep sequencing, then do a qPCR to accurately determine the number of indexing cycles required in order to limit PCR duplicates in your library and retain as much complexity as possible.Clean


After the index PCR, clean the indexed libraries using the SPRI bead cleanup protocol, in the modern lab.
Protocol
NAME

Clean up of Index PCRs using SPRI Beads

CREATED BY
Vanssy Li