For the initial training of appreci8 and its subsequent evaluation, we only consider data sets with validated mutations. Validation was achieved using three different approaches: (i) a selected set of calls (mutations, polymorphisms and artifacts) was validated using Sanger sequencing. However, as variants with a VAF below 20% are difficult to confirm with this sequencing technique, (ii) we re-analyzed a subset samples by the same or another technique as validation. Six samples were re-analyzed on Illumina NextSeq. Nine samples were re-analyzed on Ion Torrent PGM (Rothberg et al., 2011 (link)). Twenty-two samples were analyzed on Roche 454 and Illumina NextSeq. NPM1 mutations were validated by LightCycler (Roche, Mannheim, Germany) based melting curve analysis (Schnittger et al., 2005 (link)). As additional validation (iii), all calls reported in case of the two training sets were manually investigated by two independent experts. The variant-specific characteristics as well as the calls themselves in the IGV (Robinson et al., 2011 (link)) were considered. In case of the five test sets, all calls categorized as true were manually investigated. Furthermore, variant-specific characteristics of all calls categorized as polymorphisms and artifacts were investigated.
Nextseq
Manufactured by Illumina
Sourced in United States, Germany, Australia, France, United Kingdom, Czechia, Canada, Switzerland, Netherlands
The NextSeq is a high-throughput sequencing system designed for a wide range of applications, including gene expression, small RNA, and targeted sequencing. It utilizes advanced sequencing-by-synthesis technology to generate high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq, by Illumina (51 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (25 mentions)
Trizol, by Thermo Fisher Scientific (22 mentions)
Rneasy mini kit, by Qiagen (22 mentions)
Nextera xt kit, by Illumina (21 mentions)
Tapestation, by Agilent Technologies (20 mentions)
Hiseq 4000, by Illumina (19 mentions)
Hiseq 2000, by Illumina (19 mentions)
Ampure xp bead, by Beckman Coulter (18 mentions)
Hiseq 2500, by Illumina (18 mentions)
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Bioanalyzer, by Agilent Technologies (15 mentions)
Nextera xt, by Illumina (14 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (10 mentions)
2100 bioanalyzer, by Agilent Technologies (10 mentions)
Dneasy blood and tissue kit, by Qiagen (10 mentions)
Kapa library quantification kit, by Roche (10 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (10 mentions)
Fragment analyzer, by Agilent Technologies (9 mentions)
Rneasy kit, by Qiagen (9 mentions)
1 155 protocols using nextseq
1
Comprehensive Validation of Genetic Variants
2
Exome Sequencing for Somatic Mutations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Agilent SureSelect Human All Exon V4 in-solution hybrid was used to capture kit to target genomic DNA region. Captured libraries are subsequently sequenced on an Illumina NextSeq aiming for ~250X coverage. Raw reads were generated according to Illumina NextSeq protocols, generating 100 bp paired end reads and subsequently aligned to the human genome reference sequence build hg19/GRCh37 using BWA followed by realignment around InDels for quality score recalibration. Single Nucleotide Variations (SNVs) were called using muTect tool (10.1038/nature12213). To obtain metastatic-specific somatic mutation calls, we performed mutation calling for metastatic cell lines, using primary samples as matched normal in Mutect run. We then used Oncotator (10.1002/humu.22771) to annotate the somatic mutations from Mutect. Finally, we performed additional filtering (using Oncotator’s annotations for Exome Sequencing Project and dbSNP databases) to further filter germline mutations. Following somatic mutation calling and filtering, we ran MutSigCV tool (10.1038/nature12213) to determine the acquired mutations. Subsequently, the top ranked genes (196 genes; p value < 0.05), were selected and submitted to The Database for Annotation, Visualization and Integrated Discovery (DAVID) for gene-pathway enrichment analysis.
3
Transcriptomic Profiling of Regenerated Tendons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Uninjured and regenerated Patellar tendons from Tppp3ECE/+;R26RtdT;ScxGFP mice were isolated by FACS, spun down, re-suspended to 1000 cells per subpopulation, and subjected to RNA Direct-zol kit (Zymo), followed by Ovation RNA-seq V2 System (NuGEN) to generate cDNAs. cDNAs were sonicated to 300-500bp (Covaris) and libraries were generated by the TruSeq RNA Library Prep Kit (Illumina) for single-end 150bp reads (Next-seq, Illumina). For uninjured tdT+H2B-eGFP− and tdT+H2B-eGFP+ (from (Tppp3CG/+;R26RtdT;PdgfraH2B-eGFPmice) subpopulations, mRNA was extracted from 800 intact cells per replicate using the SMART-seq v4 Ultra Low Input Kit (Clontech). Amplified cDNAs were sonicated to 300-500bp (Covaris) and libraries were generated by the ThruPLEX™DNA-seq Kit (Rubicon Genomics) for single-end 150bp reads (Next-seq, Illumina). All reads were trimmed to 100bp for comparative purposes. RNA-seq data have been submitted to NCBI GEO (SUB4050561).
4
Transcriptomic Profiling of Regenerated Tendons
5
Sequencing and Typing of Enteroviruses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Original stool samples and their respective clinical isolates of CVA-13, CVA-20, and EV-C99 were sequenced using a previously published protocol [40 (link)]. Briefly, MagNA Pure 96 (Roche Diagnostics, Basel, Switzerland) was used for RNA extraction; Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) for tagmentation and library preparation; and Nextseq (Illumina, San Diego, USA) for the sequencing runs. Samples cultured directly on HAE were sequenced using MagNA Pure 96 (Roche Diagnostics, Basel, Switzerland) for DNA/RNA extraction, DNase treatment, and RNA concentration with RNA Clean & Concentrator™-5 (Zymo research, Irvine, USA). The Illumina DNA Prep kit (Illumina, San Diego, USA) was used for tagmentation and library preparation and Nextseq (Illumina, San Diego, USA) for the sequencing runs. The full-length sequences were obtained using the Jovian platform (https://jovian.rivm-bioinformatics.com , web based) and Genome Detective v1.139 (https://www.genomedetective.com/app/typingtool/virus/ , web-based). The obtained full-length sequences were aligned using CodonCode Aligner software version 9.0.2 (CodonCode Corporation, Centerville, OH, USA). Based on the complete sequence of the VP1 region for all three viruses, the typing of the original fecal material and the clinical isolates was confirmed.
6
ChIP-seq and RNA-seq of Primary Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Details for ChIP-sequencing (ChIPseq) and RNA-sequencing (RNAseq), including sample preparation, ERCC Spike-ins, and data analysis, can be found in the Supplemental material . Briefly, ChIPseq libraries were prepared at the end of primary passage. Cells were fixed followed by chromatin shearing and antibody-mediated isolation. Antibodies used can be found in Supplemental Table S2 . Libraries were created using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S). Sequencing was performed on an Illumina NextSeq (single-end, 75 cycles). RNA libraries were also prepared at the end of primary passage. After an ERCC-spike in, libraries were prepared using the NEB Ultra RNA library Prep Kit (#E7350). Sequencing was performed on an Illumina NextSeq (paired-end, 38 cycles each). Data can be found in GEO (Accession # GSE140361).
7
Optimized High-Throughput Protein Screening
8
Microbial Community Profiling via Illumina Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Library Preparation and Sequencing: The bacterial community composition was determined by Illumina NextSeq-based high-throughput sequencing (HTS) of the 16S rRNA gene V3-region, according to Krych and colleagues. (2018) [46 (link)] and is described in detail in the supplementary materials and methods . Briefly, the amplified fragments with adapters and tags were purified and normalized using custom made beads, pooled, and subjected to 150 bp pair-ended Illumina NextSeq (V3 region 16S rRNA) sequencing. The raw dataset containing pair-end reads with corresponding quality scores were merged and trimmed, followed by de-replication, purging of chimeric reads, and constructing high quality (97% similarity level) Operational Toxonomic Unit (OTU) that and taxonomically assigned using sintax coupled to the EZtaxon 16S rRNA gene reference database. The sequencing dept. was on average 68,194 read per sample before filtering going down to 62,426 after filtering.
Sequencing data pre-processing: The dataset was purged for OTU’s which were detected below 0.005% across all samples, and normalization was accomplished using MetagenomeSeq v 1.32.0 based on Cumulative Sum Scaling algorithm [47 (link)].
Sequencing data pre-processing: The dataset was purged for OTU’s which were detected below 0.005% across all samples, and normalization was accomplished using MetagenomeSeq v 1.32.0 based on Cumulative Sum Scaling algorithm [47 (link)].
9
Single-Cell RNA-Seq Protocol for Epithelial and Lamina Propria
10
Single-Cell RNA-Seq Protocol for Epithelial and Lamina Propria
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!