Jun 14, 2017

Public workspaceIllumina library construction Protocol

GigaScience
DOI
dx.doi.org/10.17504/protocols.io.iajcacn
  • SARAH SIU TZE MAK1,
  • SHYAM GOPALAKRISHNAN1,
  • CHRISTIAN CAROE1,
  • CHUNYU GENG1,
  • SHANLIN LIU1,
  • MIKKEL-HOLGER S SINDING1,
  • LUKAS F K KUDERNA1,
  • WENWEI ZHANG1,
  • SHUJIN FU1,
  • FILIPE G VIEIRA1,
  • MIETJE GERMONPRÉ1,
  • HERVÉ BOCHERENS1,
  • SERGEY FEDOROV1,
  • BENT PETERSEN1,
  • THOMAS SICHERITZ-PONTEN1,
  • TOMAS MARQUES-BONET1,
  • GUOJIE ZHANG1,
  • HUI JIANG1,
  • M THOMAS P GILBERT1
  • 1UNIVERSITY OF COPENHAGEN
  • GigaScience Press
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DOI: dx.doi.org/10.17504/protocols.io.iajcacn
External link: https://doi.org/10.1093/gigascience/gix049
Protocol Citation: SARAH SIU TZE MAK, SHYAM GOPALAKRISHNAN, CHRISTIAN CAROE, CHUNYU GENG, SHANLIN LIU, MIKKEL-HOLGER S SINDING, LUKAS F K KUDERNA, WENWEI ZHANG, SHUJIN FU, FILIPE G VIEIRA, MIETJE GERMONPRÉ, HERVÉ BOCHERENS, SERGEY FEDOROV, BENT PETERSEN, THOMAS SICHERITZ-PONTEN, TOMAS MARQUES-BONET, GUOJIE ZHANG, HUI JIANG, M THOMAS P GILBERT 2017. Illumina library construction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.iajcacn
Manuscript citation:
Mak SST, Gopalakrishnan S, Carøe C, Geng C, Liu S, Sinding MS, Kuderna LF, Zhang W, Fu S, Vieira FG, Germonpré M, Bocherens H, Fedorov S, Petersen B, Sicheritz-Pontén T, Marques-Bonet T, Zhang G, Jiang H, Gilbert MTP, Comparative performance of the BGISEQ-500 vs Illumina HiSeq2500 sequencing platforms for palaeogenomic sequencing. GigaScience 6(8). doi: 10.1093/gigascience/gix049
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
Created: June 02, 2017
Last Modified: March 27, 2018
Protocol Integer ID: 6187
Abstract
This single-tube library construction protocol is for degraded DNA using adapters for the Illumina platform.
Materials
MATERIALS
ReagentBovine Serum Albumin
Reagent25% Poly-Ethylene Glycol
ReagentNaCl
ReagentPB binding bufferQiagenCatalog #19066
ReagentMonarch DNA Cleanup Columns (5ug) - 100 columnsNew England BiolabsCatalog #T1034L
ReagentBuffer PEQiagenCatalog #19065
ReagentBuffer EBQiagenCatalog #19086
Reagent0.2 mM dNTPsThermo Fisher ScientificCatalog #AM8200
ReagentAmpliTaq Gold DNA polymerase Applied Biosystems (ThermoFisher Scientific)
ReagentMgCl2Applied Biosystems (ThermoFisher Scientific)
ReagentGeneAmp® 10X PCR Buffer II Applied Biosystems (ThermoFisher Scientific)
ReagentSYBR GreenThermo Fisher Scientific
ReagentIS7 and IS8 primers following Meyer and Kircher 2010
ReagentAccuGene molecular biology water LonzaCatalog #51200
ReagentBSA-Molecular Biology Grade - 12 mgNew England BiolabsCatalog #B9000S
ReagentIllumina InPE 1.0 forward
Reagentcustom made reverse primers
Before start
Calculate the DNA input and prepare the library adapters.
End-repair
End-repair
End-repair step following Carøe et al. 2017 with the addition of 1 μl "Reaction enhancer" consisting of 25% Poly-Ethylene Glycol (PEG4000); 2 μg/μL Bovine Serum Albumin (BSA) and 400 mM NaCl.
Note
Carøe C, Gopalakrishnan S, Vinner L, Mak SST, Sindin MHS, Samaniego JA, et al. Single-tube library preparation for degraded DNA. Methods in ecology and evolution; 2017; in press.
Ligation
Ligation
Ligation step following Carøe et al. 2017 with addition of 1 μl 10 μM adapters suited for the Illumina platform.
Note
Carøe C, Gopalakrishnan S, Vinner L, Mak SST, Sindin MHS, Samaniego JA, et al. Single-tube library preparation for degraded DNA. Methods in ecology and evolution; 2017; in press.
Adpater Fill-in & library purification
Adpater Fill-in & library purification
Fill-in step of the adapter to complete library building and purification using 1:5 volume of PB binding buffer (Qiagen) and using Monarch DNA Cleanup Columns (New England Biolabs, Massachusetts, USA).
Note
Carøe C, Gopalakrishnan S, Vinner L, Mak SST, Sindin MHS, Samaniego JA, et al. Single-tube library preparation for degraded DNA. Methods in ecology and evolution; 2017; in press.
Wash with 750 µl buffer PE (Qiagen).
Incubate for 5 minutes at 37 °C.
Duration00:05:00
Elute in 40 µl buffer EB (Qiagen). 
Quantitative real-time PCR (qPCR)
Quantitative real-time PCR (qPCR)
To determine the number of cycles used in index PCR, perform a qPCR in a 20 µl reaction volume using 1:20 dilution of:
  • purified library template
  • 0.2 mM dNTPs (Invitrogen)
  • 0.04 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, California, USA)
  • 2.5 mM MgCl2 (Applied Biosystems)
  • 1X GeneAmp® 10X PCR Buffer II (Applied Biosystems)
  • 1 µl SYBR Green (Invitrogen, Carlsbad, California, USA)
  • 0.2 µM forward and reverse primers mixture (IS7 and IS8 primers following Meyer and Kircher 2010)
  • 13.48 µl AccuGene molecular biology water (Lonza) 
qPCR cycling conditions:
cyclestempraturetime
195 °C10 min
40 95 °C30 sec
60 °C60 sec
72 °C60 sec
(using the Agilent MX3005 qPCR machine)
PCR amplification
PCR amplification
100 µl PCR reactions containing:
  • 20 µl of purified library
  • 0.2 mM dNTPs (Invitrogen)
  • 0.1 U/µl AmpliTaq Gold DNA polymerase (Applied Biosystems)
  • 2.5 mM MgCl2 (Applied Biosystems)
  • 1X GeneAmp® 10X PCR Buffer II (Applied Biosystems)
  • 0.4 mg/ml BSA (New England Biolabs Inc)
  • 0.2 µM of each forward (Illumina InPE 1.0 forward)
  • custom made reverse primers
  • 51.2 µl AccuGene molecular biology water (Lonza, Basel, CH) 
PCR cycling conditions:
cyclestempraturetime
initial denaturation95 °C12 min
13-2195 °C30 sec
60 °C30 sec
72 °C40 sec
elongation step72 °C5 min
Post-PCR, purify libraries with QiaQuick columns (Qiagen). 
Incubate libraries for 10 min at 37 °C.
Duration00:10:00
Elute libraries with 30 µl buffer EB (Qiagen). 
Bead purification
Bead purification
Use small amounts of aliquots after purification for concentration quantification and fragment size estimation with the High-Sensitivity DNA Assay kit 2100 expert High Sensitivity DNA Assay in Bioanalyzer 2100 (Agilent). 
Final purification using the AMpure XP system (Agentcourt, Beckman Counter, Indianapolis, USA) with 1.8X beads:library ratio, in order to remove any persisting primer dimers or other molecules with a fragment size of <100 bp.
Quantification
Quantification
Prior to sequencing submission, use small amounts of aliquots after purification concentration quantification and fragment size estimation with the High-Sensitivity DNA Assay kit 2100 expert High Sensitivity DNA Assay in Bioanalyzer 2100 (Agilent). 
Pool libraries in equimolar concentrations for sequencing.
Note
Meyer M, Kircher M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. 2010;2010: db.prot5448.