Truseq dna pcr free protocol

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The TruSeq DNA PCR Free protocol is a library preparation kit designed for high-quality sequencing of DNA samples without the need for PCR amplification. The protocol provides a streamlined workflow that simplifies library construction and reduces potential biases introduced by PCR.

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TruSeq protocol (148 citations) TruSeq PCR (39 citations) TruSeq DNA PCR-Free (27 citations) TruSeq DNA PCR (18 citations) TruSeq DNA protocol (17 citations) TruSeq PCR free protocol (7 citations) DNA PCR-free protocol (2 citations) TruSeq DNA PCR-free LT Library Preparation protocol (2 citations) TruSeq DNA PCR-Free Library Prep Protocol (2 citations)

8 protocols using truseq dna pcr free protocol

Genomic DNA Sequencing and Annotation

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Genomic DNA was extracted using the Wizard^® Genomic DNA Purification Kit (Promega, Madison, WI, USA). Quantity and quality were evaluated using a Qubit fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were prepared using the TruSeq DNA PCR Free protocol (Ilumina, San Diego, CA, USA). Then, the final libraries quality was assessed with Fragment Analyzer (Std. Sens. NGS Fragment Analysis kit 1- 6000 bp), and quantified by qPCR at the Genomics and Bioinformatics Core Facility (CIBIR). Subsequent sequencing was carried out in an Illumina HiSeq 1500 (Illumina, San Diego, CA, USA).
FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to analyze the quality of raw reads, which were subsequently trimmed and filtered by using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Genomes were reconstructed using PLACNETw [37 (link)] or PATRIC [38 (link)]. Identification of Open Reading Frames (ORFs) and genome annotation of the assembled genetic elements was performed by PROKKA v1.13 [39 (link)].
The genomes of P. aeruginosa PAO1, UCBPP-PA14 and PA7 strains, and the pUM505 plasmid sequence were downloaded from the NCBI database (GenBank accession No. GCF_000006765.1, GCF_000014625.1, GCF_000017205.1 and HM560971, respectively).

López M., Rojo-Bezares B., Chichón G, & Sáenz Y. (2022). Resistance to Fluoroquinolones in Pseudomonas aeruginosa from Human, Animal, Food and Environmental Origin: The Role of CrpP and Mobilizable ICEs. Antibiotics, 11(9), 1271.

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Paired-end sequencing of genomic and transcriptomic libraries

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Genomic libraries (paired-end, 150 bp) for sequencing were prepared with the Illumina TruSeq DNA PCR-Free protocol and sequenced on two Illumina Novaseq 6000 S4 flowcells. Paired-end total RNA libraries (150 bp) were prepared with the TruSeq Stranded Total RNA protocol and included rRNA depletion with Ribo-Zero Plus. Libraries were sequenced on four S4 flowcells and one S2 flowcell of the Illumina Novaseq 6000.

Mapel X.M., Kadri N.K., Leonard A.S., He Q., Lloret-Villas A., Bhati M., Hiltpold M, & Pausch H. (2024). Molecular quantitative trait loci in reproductive tissues impact male fertility in cattle. Nature Communications, 15, 674.

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Whole-Genome Sequencing of Canines

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Whole-genome sequences were generated for 51 individuals belonging to closed-breed populations using the Illumina TruSeq DNA PCR-Free Protocol (Cat.# FC-121-3001). Libraries were constructed with fragments of 300–500 bp and sequenced on the Illumina HiSeq 2000 platform using 100-bp paired-end parameters. To fully explore the documented variation in canines, sequences for all canine genomes available at the onset of this study that were sequenced on a contemporary Illumina platform were also obtained via BioProject accessions from the Sequence Read Archive or the European Nucleotide Archive (Supplemental Table S1).

Decker B., Davis B.W., Rimbault M., Long A.H., Karlins E., Jagannathan V., Reiman R., Parker H.G., Drögemüller C., Corneveaux J.J., Chapman E.S., Trent J.M., Leeb T., Huentelman M.J., Wayne R.K., Karyadi D.M, & Ostrander E.A. (2015). Comparison against 186 canid whole-genome sequences reveals survival strategies of an ancient clonally transmissible canine tumor. Genome Research, 25(11), 1646-1655.

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Illumina NovaSeq6000 Paired-end Sequencing

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The genomic DNA libraries were prepared following the TruSeq DNA PCR-free protocol (Illumina, CA, United States). A minimum of 1 µg of genomic DNA was sheared by sonication and then purified. Oligonucleotide adaptors to sequence both ends were ligated on end-repaired fragments and then purified. DNA libraries were barcoded (indexed) and then multiplexed. GS was performed at the Centre National de Recherche en Génomique Humaine (CNRGH, CEA) using the Illumina NovaSeq6000 platform (Illumina, CA, United States), generating 150 base pairs paired-end reads. Data sequencing was required to meet minimum quality standards, with an average of over ×35 depth of coverage and more than 97% of the genome covered by at least 10 reads.

Colin E., Duffourd Y., Tisserant E., Relator R., Bruel A.L., Tran Mau-Them F., Denommé-Pichon A.S., Safraou H., Delanne J., Jean-Marçais N., Keren B., Isidor B., Vincent M., Mignot C., Heron D., Afenjar A., Heide S., Faudet A., Charles P., Odent S., Herenger Y., Sorlin A., Moutton S., Kerkhof J., McConkey H., Chevarin M., Poë C., Couturier V., Bourgeois V., Callier P., Boland A., Olaso R., Philippe C., Sadikovic B., Thauvin-Robinet C., Faivre L., Deleuze J.F, & Vitobello A. (2022). OMIXCARE: OMICS technologies solved about 33% of the patients with heterogeneous rare neuro-developmental disorders and negative exome sequencing results and identified 13% additional candidate variants. Frontiers in Cell and Developmental Biology, 10, 1021785.

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Sequencing of Hot and Cold Evolved Drosophila

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We previously sequenced the hot evolved populations until generation 60 and the cold evolved populations until generation 40. All populations were sequenced as pools (Pool-Seq Schlötterer et al. 2014 (link)) at each 10th generation using the Illumina paired-end technology (Accession numbers PRJEB20533 and PRJEB20780).
Here we additionally sequenced the cold evolved populations at each 10th generation from generation 50 to 100. For each sample the DNA of pooled female and male flies was extracted using a high salt extraction protocol (Miller et al. 1988 (link)) and sheared with a Covaris S2 device (Covaris, Inc. Woburn, MA, USA). Libraries were prepared using the TruSeq DNA PCR-Free protocol (Illumina, San Diego, CA) and sequencing was performed with the Illumina HiSeq X Ten platform (Illumina, San Diego, CA). For an overview of the sequencing data used in this work see supplementary table S1, Supplementary Material online.

Kofler R., Nolte V, & Schlötterer C. (2022). The Transposition Rate Has Little Influence on the Plateauing Level of the P-element. Molecular Biology and Evolution, 39(7), msac141.

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Extracting and Sequencing Gerbil Liver DNA

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DNA from gerbil livers was extracted using Qiagen Blood and Tissue DNeasy kit (Qiagen Inc., USA) following the manufacture’s protocol. DNA extractions were freeze dried prior to shipping to the University of Oslo and upon arrival resuspended in 200 μl E.Z.N.A. Elution buffer (Omega Biotek) and placed in a heating block at 37°C for 4 h. The extractions were then analyzed with Qubit (Thermo Fisher Scientific), NanoDrop (Thermo Fisher Scientific), and Bioanalyzer (2100, Agilent Technologies) to assess the quality and quantity of DNA. DNA samples from ten survivors and ten moribund individuals (n = 20) from the experiment, were selected for DNA sequencing based on high-quality DNA (Table 1). Prior to library prep, an additional 100 µl E.Z.N.A. Elution buffer was added to the samples due to high DNA yields. Library prep was performed using the Illumina TruSeq DNA PCR Free protocol and the samples were sequenced using Illumina HighSeq 2500 with a 350 bp insert size at the Norwegian Sequencing Centre (NSC), University of Oslo, Norway.

Nilsson P., Ravinet M., Cui Y., Berg P.R., Zhang Y., Guo R., Luo T., Song Y., Trucchi E., Hoff S.N., Lv R., Schmid B.V., Easterday W.R., Jakobsen K.S., Stenseth N.C., Yang R, & Jentoft S. (2022). Polygenic plague resistance in the great gerbil uncovered by population sequencing. PNAS Nexus, 1(5), pgac211.

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Genome Sequencing of Commercial Pigs

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Twenty-six commercial pigs were used in this study. Tissue samples were collected from ear punches or tail clippings and genomic DNA was extracted using Qiagen DNeasy 96 Blood & Tissue kits (Qiagen Ltd., Mississauga, ON, Canada). Paired-end library preparation was conducted using the TruSeq DNA PCR-free protocol (Illumina, San Diego, CA). Two sets of libraries were produced; one with an average insert size of 350 bp and the other with an average insert size of 550 bp. Libraries with an average insert size of 350 bp were sequenced on a HiSeq 4000 instrument (Illumina, San Diego, CA), for a target coverage of 2× per sample. For this, all 26 samples were multiplexed within a single flow cell channel. Libraries with an average insert size of 550 bp were sequenced on a HiSeq X instrument (Illumina, San Diego, CA), for a target coverage of 30× per sample. For this, the 26 samples were sequenced, one sample per flow cell channel. All libraries were sequenced at Edinburgh Genomics (Edinburgh Genomics, University of Edinburgh, Edinburgh, UK). DNA samples from the same pigs were also genotyped using the GGP-Porcine HD BeadChip (GeneSeek, Lincoln, NE).

Ros-Freixedes R., Battagin M., Johnsson M., Gorjanc G., Mileham A.J., Rounsley S.D, & Hickey J.M. (2018). Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing. Genetics, Selection, Evolution : GSE, 50, 64.

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Paired-end Genomic Sequencing of Stilt and Avocet

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Paired-end libraries for the kakī and pied stilt were prepared at the University of Otago using the Illumina TruSeq^® DNA PCR-free protocol according to manufacturer specifications, with genomic DNA fragmented to 350 bp. End repair and adapter ligation for sequence barcoding were carried out and libraries were indexed with unique 6 bp sequences. Sequencing of kakī and pied stilt libraries was completed by New Zealand Genomics Limited (NZGL), where sample libraries were pooled with three additional stilt samples and spread across five lanes of a flow cell for 2 × 125 bp sequencing on an Illumina HiSeq 2500.
Paired-end libraries for the pied avocet were prepared using the TruSeq^® DNA Nano Library Prep protocol according to manufacturer specifications, with genomic DNA fragmented to 350 bp. Library preparation and sequencing for the pied avocet was completed at IKMB using one lane of a flow cell on an Illumina HiSeq 4000 for 2 × 150 bp sequencing.

Galla S.J., Forsdick N.J., Brown L., Hoeppner M.P., Knapp M., Maloney R.F., Moraga R., Santure A.W, & Steeves T.E. (2018). Reference Genomes from Distantly Related Species Can Be Used for Discovery of Single Nucleotide Polymorphisms to Inform Conservation Management. Genes, 10(1), 9.

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