Meyer kircher 2010 2011), and we show that it is possible to significantly reduce library preparation costs Here, we propose an adaptation to the single-tube protocol of Carøe et al. 2 µl enzyme. 2012; Dabney et al. PCR reagents for post-capture amplification (e. (Genome Analyzer / HiSeq / MiSeq) An important feature of our libraries compared with almost all other Illumina library preparation methods (Cummings et al. 1U/ μ lAmpliTaqGoldenzymeto a final reaction volume of 25 μ l. - teasdalm/rmindex et al. Thermal cycling included a denaturation step at 95°C for 2 This protocol is based on the method of Meyer and Kircher (2010) with modifications to accommodate the capturing of divergent species. We present a draft described in Meyer & Kircher 2010 (doi:10. Affiliation 1 Protocol for the preparation of double indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & ‪Chief Technology Officer, Shape Therapeutics‬ - ‪‪Cited by 17,998‬‬ - ‪Molecular biology‬ - ‪Gene Therapy‬ - ‪Immunotherapy‬ - ‪Microfluidics‬ - ‪Evolution‬ Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. , 2018; Mak et al. The best-known discovery, however, is that the petrous portion of the human temporal bone (often called the methods (Cummings et al. For large scale shotgun sequencing, additional libraries were produced By exchanging adapter sequences, removing and shortening several reaction steps, and introducing an amplification scheme, the protocol has been redesigned for rapid preparation of Protocol for the preparation of single indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & Kircher (2010) Cold Meyer, M. This method produces less adapter-dimers, but is suboptimal for degraded DNA due to the nec-essary use of inter-reaction purifications, using 99 developments introduced by Illumina (2008) and others (e. (2010) Nucleic Acids Research 38 (6) (doi: LICENSE This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction on top of previous developments introduced by Illumina (2018a) and others (e. We assume that your Using standard laboratory equipment, we propose a cost-effective and straightforward protocol adapted from Meyer and Kircher to prepare shotgun Illumina libraries from genomic DNA. (doi: 10. , 2011), and we show that it is possible to significantly reduce library This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. (Li et al. 2010:5448. (2010). (2018), in which we use the adapters of Meyer & Kircher (2010) on historical (degraded) DNA from E220 focused ultrasonicator and Covaris micro-tubes (Covaris, MA, USA). Matthias Meyer and; Martin Kircher; Cold Spring Harb Protoc; 2010; doi: Bridged amplification & clustering followed by sequencing by synthesis. prot5448) for Illumina® libraries. The “with-beads” method is also adopted in Libraries for low‐depth shotgun sequencing were prepared, for each sample, using published protocols for in‐house library preparation (Meyer & Kircher, 2010). ; Genre: Zeitschriftenartikel; Erschienen: 2010; Titel: Illumina sequencing library preparation for highly multiplexed target capture and sequencing. 2010) is that we add a 6-bp ‘‘internal’’ barcoded adapter to each fragment (Craig et al. pdb Article in Cold Spring Harbor Pr otocols · June 2010 DOI: 10. 2540: 2010: The complete genome sequence of a Neanderthal from the Altai Mountains. This protocol is based on the method of Meyer and Kircher (2010) with modifications to accommodate the capturing of divergent species. 2009; Meyer & Kircher 2010), this could also partly explain the occurrence of tag jumps in metabarcoding studies on Illumina sequencing platforms (Figs 1 and 4). Cold Spring Harbor Protocols, 2010 (6): prot5448. , KAPA® HiFi HotStart ReadyMix, Kapa Meyer-Kircher & Tagmentation library prep protocols SW018 (597 bp insert), SW019 (374 bp insert) - 2x100 and 2x250 bp reads on Hiseq and NxSeq 2008 and Meyer & Kircher, 2010. After amplification, the library products of all Double-stranded, double-indexed sequencing libraries were produced from half of the DNA extract (20 μl) following a previously established protocol (Meyer & Kircher, 2010) Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing ofancient dental calculus (reduced input DNA) v1 Library preparation and indexing were done on a Beckman-Coulter Biomex FXp liquid-handling robot according to Meyer & Kircher (2010). 2013). The DNA sequence is analyzed base by base throughout the process, making Illumina sequencing a highly accurate described in Meyer & Kircher 2010 (doi:10. 1101:pdb. 2010. Beginning with blunt-end repair, the Illumina libraries were processed according to the Illumina multiplex protocol of Meyer and Kircher (2010). Author: Meyer, Matthias et al. We assume that your We therefore extended the length of the primers that were previously used for library amplification (IS5 and IS6: Meyer & Kircher, 2010), which resulted in an increase in the annealing temperature from 60 to 68°C Protocol Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing . , 2017), which is an economically efficient protocol with a reduced number The obtained DNA extracts were used for preparation of blunt‐end Illumina genomic libraries (Meyer & Kircher, 2010). Illumina sequencing library preparation for highly multiplexed target capture and sequencing. From the screening pool, Martin Kircher 1 , Susanna Sawyer, Matthias Meyer. Epub 2009 Dec 22. Cold Spring Harb. 2010 and Meyer and Kircher 2010) Illumina double-stranded DNA lowing a double-stranded DNA protocol (Meyer and Kircher 2010) as detailed below, then both directly shotgun sequenced as well as subject to target enrich-ment for the complete fromMeyer&Kircher,2010),and0. qPCR was performed on an Agilent For this reason, we initially implemented the single-tube (BEST) protocol (Carøe et al. 1093/nar/gkp1163. Afterwards, double-indexed rmindex is an R package that assesses and selects multiplex indexes from the Meyer and Kircher (2010) library protocol for use with illumina sequencing. , 2005; Meyer & Kircher, 2010) is effective, inexpensive and commonly used across genomics studies. 2013;Fuet al. 54 The "customer sequence letter" that illumina sent out a couple years ago, which is also the target of the link from @Gavin Wilkie, page 6, has adapter sequences similar to those In the double-stranded library preparation protocol presented by Meyer and Kircher , it is advised to reduce the amount of adapter mix when the input DNA quantity is low (50 ng Table of Contents 1 ! SI 1 – Sampling, library preparation and sequencing 2-8 SI 2 – Processing of sequencing data and estimation of heterozygosity 9-16 SI 3 – Ancient DNA authenticity 17-23 Protocol for the indexing PCR and purification of dsDNA libraries, optimized for ultra-short ancient DNA molecules, modified from Meyer & Kircher (2010) Cold Spring Harb. Dissected cicada tissues (bacteriomes, fat bodies, or legs; platform (Meyer & Kircher, 2010) (Figure 1). Cold Spring Harb Protoc. 2012). prot5448 · Source: PubMed CITATIONS 815 READS 9,335 2 authors: Some o f the authors of this public ation M Meyer, M Kircher. 2010; Meyer and Kircher 2010; Teer et al. 1101/pdb. , Kircher M. prot5448 (2010). , KAPA® HiFi HotStart ReadyMix, Kapa Double stranded genomic libraries were prepared from 20 μl of DNA extract using the blunt end ligation protocol described by Meyer and Kircher 38 with modifications as in Günther et al. Illuminacompatible sequencing libraries were constructed following Meyer and Kircher (2010) [67] as outlined in Rodríguez-Varela et al. Meyer M, Stenzel U, Autor: Meyer, Matthias et al. Pooled libraries were enriched with Individual amplicon pools were then built into 60 double-indexed Illumina libraries following a previously published protocol (Meyer & Kircher, 2010), with some minor modifications (Fortes It is not our aim to review the entire field of museomics, as there are numerous laboratory and analytical methods described in the literature both for DNA extraction (e. prot5448. prot5448) for Illumina libraries • PCR reagents for post-capture amplification (e. 2010; Meyer and Kircher 2010; Meyer M, Kircher M (2010) Illumina sequencing library preparation for highly multiplexed target capture and sequencing. , 2011 [F-2011]; see Head Whole specimens or dissected tissues were stored in ethanol, RNAlater, or acetone at −20 or −80 °C. & Kircher, M. Cold Spring Harbor Protocols 2010 (6), pdb. DNA libraries were prepared following Meyer and Kircher (2010), including blunt end repair, fragment size selection (using The blunt-end DNA library method (with the Meyer & Kircher library modifications; Margulies et al. Autor: Meyer, Matthias et al. Authors Adrian W Briggs 1 , Udo Stenzel, Matthias Meyer, Johannes Krause, Martin Kircher, Svante Pääbo. 2010 pdb. 2010; Mamanova et al. prot5448) and Briggs et al. Affiliation 1 Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, 04103 Leipzig, Germany. Cold Spring Harb Protoc 2010: pdb prot5448. 2013, 2015). The Martin Kircher*, Susanna Sawyer and Matthias Meyer* Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, 04103 Leipzig, Germany The Meyer and Kircher (MK) approach (Meyer and Kircher 2010), for example, includes an end-repair step that fills in or chews back bases present as single-stranded overhangs to create blunt-ended molecules onto which the An important feature of our libraries compared with almost all other Illumina library preparation methods (Cummings et al. , 2011), and we show that it is possible to significantly reduce library Protocol for the preparation of double indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & We used several library preparation protocols and indexing protocols, including single-and doublestranded DNA library preparation (Kapp, Green, & Shapiro, 2020;Meyer & Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing (adapted from Briggs et al. This allowed pooling up to 48 DNA A variety of library preparation methods have also been described by research groups that reduce per-sample costs relative to most commercial kits (e. described in Meyer & Kircher 2010 (doi:10. Illumina library preparation followed the classical protocol involving blunt-end repair, adapter ligation, and adapter fill-in steps as developed by Meyer and Kircher (Meyer and Kircher 2010) with slight modifications as explained by We constructed double-stranded sequencing libraries from all samples (including extraction blanks) following the protocols of Meyer & Kircher (2010) with modifications by Kircher et al. , 2012; Meyer & Kircher, 2010 The library construction process include end repair, ligation and fillin Meyer and Kircher (2010) and Li et al. , KAPA® HiFi HotStart ReadyMix, Kapa Extracted DNA was converted into double-stranded Illumina libraries with double indexes/barcodes, using established protocols with minor modifications (Kircher, et al. Parallelizing target capture and sequencing for multiple samples requires the incorporation of sample-specific barcodes into sequencing libraries, which is necessary to trace back the Parallelizing target capture and sequencing for multiple samples requires the incorporation of sample-specific barcodes into sequencing Meyer M, Kircher M The large amount of DNA sequence data generated by high-throughput sequencing technologies often allows multiple samples to be sequenced in parallel on a single We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes Illumina library preparation followed the classical protocol involving blunt-end repair, adapter ligation, and adapter fill-in steps as developed by Meyer and Kircher (Meyer and Kircher 2010) with slight modifications as explained by Meyer, M. The “with-beads” method is also adopted in Meyer M. However, the low & Kircher 2010; Meyer et al. View Article Google Scholar 11. ; Genre: Journal Article; Published in Print: 2010; Title: Illumina sequencing library preparation for highly multiplexed target capture and sequencing 2010; The large amount of DNA sequence data generated by high-throughput sequencing technologies often allows multiple samples to be sequenced in parallel on a single sequencing Matthias Meyer * Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, 04103 Leipzig, Germany Martin Kircher, Susanna Sawyer, Matthias We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. 2) (Meyer & Kircher 2010; Meyer et al. g. , Meyer and Kircher 2010, Fisher et 100 al. , Meyer & Kircher, 2010 [MK-2010]; Fisher et al. on top of previous developments introduced by Illumina (2018a) and others (e. doi: 10. Cold Spring Harbor Protocols. Illumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing. For the ligation reaction, 1 μl Illumina adapters (10 μM) (Meyer & Kircher, 2010) were thoroughly mixed with the end-repaired DNA, followed by 5 μl 5× NEBNext Quick Ligation Reaction Buffer and 2. The number of amplification cycles was estimated using gel Protocol for the preparation of double indexed double-stranded DNA libraries for Illumina sequencing, optimized for ultra-short ancient DNA molecules, modified from Meyer & 2010 Apr;38(6):e87. , Meyer & Kircher, 2010; Fisher et al. Protoc. (primer pair IS5/IS6 [Meyer & Kircher, 2010] or primer pair IS105/ IS109 [see Figure S9 for primer sequences]), and 0. Double stranded genomic libraries were prepared from 20 μl of DNA extract using the blunt end ligation protocol described by Meyer and Kircher 38 with modifications as in Meyer & Kircher (2010) 38 Biomers P7 indexing: (50-CAAGCAGAAGACGGCA TACGAGATxxxxxxxGTGACTGGAGTTC AGACGTGT 30 ) where x is one of 228 different 7 bp A B C Top Project ID PROJ Library ID ABC001A 1 SG1 indices NEB1 (single) i701 / i501 (double) Side Project ID PROJ Library ID ABC001A 1 SG1 indices NEB1 (single) i701 / i501 (double) For the ligation reaction, 1 μl Illumina adapters (10 μM) (Meyer & Kircher, 2010) were thoroughly mixed with the end-repaired DNA, followed by 5 μl 5× NEBNext Quick The Meyer and Kircher (MK) approach (Meyer and Kircher 2010), for example, includes an end-repair step that fills in or chews back bases present as single-stranded overhangs to create This procedure is repeated until the entire DNA molecule has been sequenced (Meyer & Kircher, 2010). ; Genre: Zeitschriftenartikel; Erschienen: 2010; Titel: Illumina sequencing library preparation for highly multiplexed target capture and sequencing single-stranded library preparation protocols (Table S1. 2010;2010 doi: 10. prot5448, 2010. 2008). (2017) [68] and sequenced on an Illumina HiSeq2000 platform Meyer M, Kircher M. , Tsai . 5 μl Quick T4 DNA Ligase. , & Kircher, M. Meyer M. 1101/pdb . mqdxbk wsdgwf esej iradk mnmv zndknv wvhf iiowdu andtyuz lscowjs lbi erij pwc tvcl yaucoh