mRNA from the five developmental stages, other than the medusa, was isolated from whole organisms (hundreds of individuals). mRNA from the medusa stage was isolated from several specimens during spring 2012, and only the middle part of the bell was extracted using Tri-Reagent Kit (Sigma). Samples were prepared for sequencing using Illumina’s TruSeq RNA Library Prep Kit (Illumina) according to the manufacturer’s instructions. Six samples were sequenced using 100-bp paired-end reads on an Illumina HiSeq2000 lane and TruSeq v3 flow chamber at the Life Sciences and Engineering Infrastructure Unit, Technion, Haifa.
Hiseq 2000 lane
Manufactured by Illumina
Sourced in United States
The HiSeq 2000 lane is a component of the HiSeq 2000 sequencing system. It is a high-throughput, massively parallel sequencing system designed for large-scale DNA and RNA sequencing applications. The HiSeq 2000 lane is responsible for accommodating and processing samples during the sequencing workflow.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Truseq rna sample prep kit, by Illumina (2 mentions)
Truseq small rna kit, by Illumina (2 mentions)
Nebnext small rna library prep set for kit, by Illumina (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Truseq stranded mrna library prep kit, by Illumina (2 mentions)
Tri reagent kit, by Merck Group (1 mentions)
Truseq rna library prep kit, by Illumina (1 mentions)
Megascript t7 kit, by Thermo Fisher Scientific (1 mentions)
Ribo zero gold rrna removal kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer rna 6000 pico chip, by Agilent Technologies (1 mentions)
Rq1 dnase 1, by Promega (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
Dneasy tissue kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Sureselectxt2 target enrichment system, by Agilent Technologies (1 mentions)
Dneasy animal blood and tissue kit, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nextseq lane, by Illumina (1 mentions)
27 protocols using hiseq 2000 lane
1
Transcriptome Profiling of Jellyfish Development
2
Illumina Sequencing Library Preparation
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DNA libraries for Illumina sequencing were generated using standard protocols. Briefly, 700ng to 2.5ug of input DNA (depending on the level of DNA degradation assessed by agarose gel electrophoresis before shearing) was sheared to a size range of 100–500 bp, and custom inline barcodes were added during adapter ligation for paired-end sequencing.
For target selection, we utilized a set of fosmid libraries that had been previously end-sequenced and mapped to a 3x draft assembly, V17e/felCat4 [40 (link)]; identity of all fosmids was verified by PCR of each end.
A detailed protocol for probe preparation, hybridization, and capture is described elsewhere (Day et al., manuscript submitted). Briefly, fosmids were pooled according to their inferred individual mass, a total of 1.5ug of input was sheared to 100–500 bp, and DNA fragments were used to prepare biotinylated RNA with a Megascript T7 kit (Ambion). After DNAse treatment and removal of unincorporated nucleotides, hybridizations were carried out in a volume of 26ul that contained 300 ng of probe and 500ng of library DNA (125ng each of 4 inline barcoded libraries). Selected DNA was recovered with magnetic beads and amplified by PCR (20 cycles using standard Illumina primers). Library 4-plex pools were sequenced as 12-plex library sets on a single Illumina HiSeq 2000 lane.
For target selection, we utilized a set of fosmid libraries that had been previously end-sequenced and mapped to a 3x draft assembly, V17e/felCat4 [40 (link)]; identity of all fosmids was verified by PCR of each end.
A detailed protocol for probe preparation, hybridization, and capture is described elsewhere (Day et al., manuscript submitted). Briefly, fosmids were pooled according to their inferred individual mass, a total of 1.5ug of input was sheared to 100–500 bp, and DNA fragments were used to prepare biotinylated RNA with a Megascript T7 kit (Ambion). After DNAse treatment and removal of unincorporated nucleotides, hybridizations were carried out in a volume of 26ul that contained 300 ng of probe and 500ng of library DNA (125ng each of 4 inline barcoded libraries). Selected DNA was recovered with magnetic beads and amplified by PCR (20 cycles using standard Illumina primers). Library 4-plex pools were sequenced as 12-plex library sets on a single Illumina HiSeq 2000 lane.
3
Genotyping-by-Sequencing Protocol for Carrot
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Genotyping-by-sequencing (GBS) was conducted following the protocol of Elshire et al. (2011) (link) and as described for carrot (Arbizu et al., 2016 (link); Iorizzo et al., 2016 (link); Ellison et al., 2017 (link)). Library construction and sequencing were performed by the University of Wisconsin-Madison Biotechnology Center (WI, United States) using half-sized reactions. Genomic DNA was digested with ApeK1, barcoded, and pooled for sequencing with 85–95 pooled samples per Illumina HiSeq 2000 lane. Samples were sequenced using single end, 100 nt reads and v3 SBS reagents (Illumina, San Diego, CA, United States).
SNPs were called using the TASSEL-GBS pipeline version 5.2.31 (Bradbury et al., 2007 (link); Glaubitz et al., 2014 (link)). Filtering was conducted in VCFtools version 0.1.14 (Danecek et al., 2011 (link)) with the following parameters: a minimum minor allele frequency of 0.1 and maximum missing data of 10% for both genotype and marker.
SNPs were called using the TASSEL-GBS pipeline version 5.2.31 (Bradbury et al., 2007 (link); Glaubitz et al., 2014 (link)). Filtering was conducted in VCFtools version 0.1.14 (Danecek et al., 2011 (link)) with the following parameters: a minimum minor allele frequency of 0.1 and maximum missing data of 10% for both genotype and marker.
4
De novo Transcriptome Assembly and Phasmavirus Detection
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An RNA-Seq library was produced for C. americanus from total RNA extracted from four pooled larvae with the RNeasy Mini Kit (Qiagen) and subsequently treated with RQ1 DNase I (Promega). Ribosomal RNA was reduced with the RiboZero Gold rRNA removal kit (Epicentre). The library was generated with the RNA ScriptSeq v2 RNA-Seq library preparation kit (Epicentre) and quantified with the Agilent 2100 Bioanalyzer RNA 6000 Pico Chip. RNA sequencing was carried out at the University at Buffalo Next Generation sequencing facility using Rapid 150-cycle paired-end sequencing on a single Illumina HiSeq 2000 lane. Due to technical error, only single end reads were obtained (approximately 33 million 150 bp reads). CLC Genomics Workbench (http://www.qiagenbioinformatics.com ) was used for de novo sequence assembly with default parameters, which allowed the software to automatically select kmer size, bubble size, and paired distances. Assembled contigs were grouped into a custom sequence database in Geneious R7.1 (Biomatters, http://www.geneious.com ) (Kearse et al. 2012 (link)) and queried with phasmavirus amino acid sequences using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990 ) algorithm tBLASTn and an expect value cutoff of 10−5.
5
Genotyping by Sequencing for Population Genetics
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For each individual examined, we extracted total DNA from vouchered tissue samples using DNeasy tissue kits (Qiagen, Valencia, California, USA) following the manufacturer’s protocol. We sent DNA extracts to the Cornell Institute of Genomic Diversity (IGD) to collect data using Genotyping by Sequencing, a RAD-Seq method (Elshire et al., 2011 (link)). Briefly, the IGD digested DNA using PstI (CTGCAG) and ligated a sample-specific indexed adapter and common adapter to resulting fragments. The IGD pooled and cleaned ligated samples using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA), amplified the pool using an 18-cycle PCR, purified the PCR product using QIAquick columns, and quantified the amplified libraries using a PicoGreen assay (Molecular Probes, Carlsbad, California, USA). Based on the PicoGreen concentrations, the IGD then combined the samples for this project with unrelated samples and ran plates of 96 samples on a 100-base pair, single-end Illumina HiSeq 2000 lane (Illumina, San Diego, California, USA).
6
ddRAD Sequencing and SNP Calling Protocol
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A genomic library was prepared following the double-digest Restriction-Associated DNA (ddRAD) protocol of Brelsford et al. (2016a (link)). Briefly, it consisted of enzyme digestion by SbfI and MseI, ligation of barcoded adapters, PCR-amplification of the resulting fragments and size-selection of PCR products between 400 and 500 bp, and was sequenced on a single Illumina HiSeq 2000 lane.
Raw reads were demultiplexed using the process_radtags module of Stacks v. 1.24 (Catchen et al. 2013 (link)) and loci were built with the denovo_map.pl pipeline, run separately for each species, with –bound_high 0.05 and other parameters left with default values. The resulting variants were then split into separate files for mapping crosses and unrelated adults. Variants were filtered with VCFtools v0.1.10 (Danecek et al. 2011 (link)), retaining genotypes with minimum read depth of 7, and retaining loci genotyped in at least 80% of individuals. We excluded the loci with minor allele frequencies below 0.1 for unrelated adults and below 0.15 for mapping cross parents and offspring. These filters resulted in data sets of 13,622 and 16,024 SNPs for unrelated adults in H. sarda and H. savignyi, respectively, and 12,509 and 11,064 SNPs for mapping crosses in H. sarda and H. savignyi, respectively.
Raw reads were demultiplexed using the process_radtags module of Stacks v. 1.24 (Catchen et al. 2013 (link)) and loci were built with the denovo_map.pl pipeline, run separately for each species, with –bound_high 0.05 and other parameters left with default values. The resulting variants were then split into separate files for mapping crosses and unrelated adults. Variants were filtered with VCFtools v0.1.10 (Danecek et al. 2011 (link)), retaining genotypes with minimum read depth of 7, and retaining loci genotyped in at least 80% of individuals. We excluded the loci with minor allele frequencies below 0.1 for unrelated adults and below 0.15 for mapping cross parents and offspring. These filters resulted in data sets of 13,622 and 16,024 SNPs for unrelated adults in H. sarda and H. savignyi, respectively, and 12,509 and 11,064 SNPs for mapping crosses in H. sarda and H. savignyi, respectively.
7
Transcriptome Analysis of Plant Leaf Samples
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Fresh young leaf samples were frozen on dry ice in the field and stored at − 80 °C until total RNA extraction. Preliminary RNA precipitation [29 (link), 30 (link)] as performed prior to total RNA extraction with the Qiagen RNeasy Plant Mini Kit protocol with DNase treatment (Qiagen, Hilden, Germany). RNA-Seq libraries with insert length 100–380 bp (mode = 170 bp) were prepared from 4 μg of RNA using an Illumina TruSeq RNA Sample Prep Kit. Each library was uniquely tagged using 12 TruSeq indexed adapters (numbers 1–12) to enable 12-plexing of samples in each Illumina HiSeq 2000 lane.
8
Exome Sequencing of Tumor and Germline DNA
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Libraries were constructed from 500 ng of unamplified tumor or germline DNA following the Illumina TruSeq DNA Sample Preparation procedure (Illumina, San Diego, CA, USA), followed by exome capture using the NimbleGen SeqCap EZ Human Exome Library v1 or v2 capture kit (Roche NimbleGen, Heidelberg, Germany). Each resulting paired-end library was sequenced on one-third of an Illumina HiSeq2000 lane using 75 bp or 100 bp reads. Library preparation and detailed summary statistics for all samples are listed in Additional file 1 : Table S2.
9
Targeted mtDNA Sequencing Protocol
10
Comparative Genomic Shotgun Sequencing
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For comparison of genome capture efficiency, DNA shotgun sequencing was conducted on two tick samples: Sh1589 and Sh834 (Additional file 1 : Table S1). Sample preparation and genomic library preparation was conducted as described without the hybrid capture step. Genomic libraries were prepared from the two whole tick DNA extracts and sequenced directly on one Illumina HiSeq 2000 lane. The mapping pipeline was identical for both shotgun and captured samples.
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