The whole genomes of 96 individual barn owls (T.alba) were used in this study (supplementary table S1 ): 94 individuals were sampled in 11 Western Palearctic localities: Canary Islands (Tenerife Island—WC), Portugal (PT), France (FR), Switzerland (CH), Denmark (DK), Serbia (SB), Greece (GR), Italy (IT), Aegean islands (AE), Cyprus (CY), and Israel (IS). In addition, one Eastern (Tyto javanica from Singapore) and one American barn owl (Tyto furcata from California) were used as outgroups. Illumina whole-genome sequences of individuals from PT, FR, CH, DK, and the outgroups were obtained from the GenBank repository (BioProject PRJNA700797 ). For the remaining 61 individuals, we followed a similar library preparation and sequencing protocol as outlined in Machado et al. (2021) (link). Briefly, genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), and individually tagged. About 100 bp TruSeq DNA PCR-free libraries (Illumina) were prepared according to manufacturer’s instructions. Whole-genome resequencing was performed on multiplexed libraries with Illumina HiSeq 2500 PE high-throughput sequencing at the Lausanne Genomic Technologies Facility (GTF, University of Lausanne, Switzerland), producing a mean number of 121,529,696 paired-end reads per individuals (2×150 bp long, SD: 21,012,431 reads).
Hiseq 2500 pe
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The HiSeq 2500 PE is a high-throughput DNA sequencing system designed for a wide range of applications. It is capable of generating sequencing data from multiple samples in parallel, with a focus on providing efficient and accurate results.
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6 protocols using hiseq 2500 pe
1
Whole-Genome Sequencing of Barn Owls
2
RNA-Seq of Fungi on Nutrient Media
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Conidia from strains grown two weeks on ½ strength corn meal agar and 1 × 106 spores were inoculated into 100 mL of both Czapek-Dox broth and SM producing media [79 (link)]. Two replicate flasks were grown for each strain. Tissue for RNA extraction was harvested at 7 days and RNA was extracted using TRIzol (Invitrogen) according to the manufactures protocol. RNA libraries were prepared using the TruSeq RNA kit and sequenced as a depth of 12 multiplexed samples per lane on the Illumina Hi-Seq 2500 PE with 100 bp reads. RNA-Seq reads were first trimmed to 75 bp to remove poor quality reads at the beginning (15 bp) and end (10 bp) of the reads and reads with an average quality score of < 20 were removed. Transcripts were assembled using Trinity [80 (link)] and input into MAKER.
3
Whole Genome Sequencing for SNP Detection
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Genomic DNA for WGS was isolated using standard techniques. Genome sequencing and analysis of SNPs were carried out by Life Sequencing S.L. (www.lifesequencing.com ) (Illumina HiSeq. 100PE, 50–100x, MAD4650) or Otogenetics corporation (www.otogenetics.com ) [Illumina HiSeq. 2500, PE 100–125, 100x, MAD4738(AR), MAD4747 (ER), MAD4751 (FR) and MAD2 (wt)]. To pick candidate SNPs to cause resistance in each strain, we selected all missense, frameshift or 5′UTR mutations that were not present in the genomes of the strains with resistance mutations that were classified to different linkage groups. We visualized WGS data with the Integrative Genomics Viewer70 (link),71 (link).
4
Soil Microbiome Profiling via 16S rRNA Sequencing
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Total genomic DNA was extracted from 1.0 g mixture of AS (3 collection points in 2017), using the Power Soil DNA Extraction Kit (Mo Bio Laboratories Inc., Carlsbad, CA, USA) as per the manufacturer’s instructions. The 16S rRNA amplicon sequencing was performed according to our previous study (Cao et al., 2018 ); briefly, sequencing was performed on a Illumina MiSeq platform at Novogene Cooperation (Beijing, China) with a HiSeq 2500 (PE250) sequencing system, using the specific primer pair of 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5 ′-GGACTACHV GGGTWTCTAAT-3′), targeting the V3–V4 hypervariable region. The data that supporting this study are openly available in the NCBI GenBank (BioProject accession number: PRJNA664798 ).
5
Gorilla Y Chromosome Assembly and Analysis
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PacBio RSII data of a flow-sorted and amplified gorilla Y chromosome, GorY-WGA (Table 1 ), was downloaded from the NCBI Short Read Archive (SRA SRX1161235). The previously published assembly of the gorilla Y chromosome and the publicly available data from the flow-sorted, whole-genome amplified and de-branched gorilla Y chromosome, GorY [4 ], were downloaded as well (GCA_001484535.2). Canu version 1.3 was used for the assembly of the PacBio reads. For comparison, the human chromosome Y assembly (NC_000024.10), HumY, was downloaded. QUAST [40 ] was used for assembly comparison and statistics. PacBio reads were mapped to HumY, GorY and the new assemblies using BLASR (> 80% identity and > 80% read coverage); Illumina HiSeq 2500 PE reads (SRA SRR2176191) were mapped using CLCBio version 8.0.2. Statistics for all mapping results were calculated in CLCBio. For calculating the contig length distribution of the GorY assembly, scaffolds were broken up and N’s were removed. The gorilla X-degenerate gene transcripts were retrieved from a previous study [23 ]. GMAP version 2017-03-17 [24 (link)] was used to align the transcripts to the assemblies.
6
Whole-genome Resequencing Protocol
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For these new 20 individuals, we followed a similar library preparation and sequencing protocol as described in Machado et al. [26 (link)]. In brief, genomic DNA was extracted using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), and individually tagged. In total, 100 bp TruSeq DNA PCR-free libraries (Illumina) were prepared according to the manufacturer's instructions. Whole-genome resequencing was performed on multiplexed libraries with Illumina HiSeq 2500 PE high-throughput sequencing at the Lausanne Genomic Technologies Facility (GTF, University of Lausanne, Switzerland).
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