The AaegL5 assembly was deposited at NCBI in June 2017 and annotated using the NCBI RefSeq Eukaryotic gene annotation pipeline56 . Evidence to support the gene predictions came from over 9 billion Illumina RNA-seq reads, 67k Pacific Biosciences IsoSeq reads, 300k ESTs, and well-supported proteins from D. melanogaster and other insects. Annotation Release 101 was made public in July 2017, and specific gene families were subjected to manual annotation and curation. Detailed descriptions of the manual annotation and curation of multigene families (hox genes, proteases, opsins and biogenic amine receptors, chemosensory receptors, and ligand-gated ion channels) are in Supplementary Methods and Discussion . See also: https://www.ncbi.nlm.nih.gov/genome/annotation_euk/Aedes_aegypti/101/
Iso seq
Manufactured by Pacific Biosciences
Sourced in United States
Iso-Seq is a long-read sequencing technology developed by Pacific Biosciences. It enables the generation of full-length, high-quality transcripts, providing comprehensive information about gene expression and alternative splicing.
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Lab products found in correlation
Rna seq, by Pacific Biosciences (2 mentions)
Rna seq reads, by Pacific Biosciences (1 mentions)
Ampure pb bead, by Pacific Biosciences (1 mentions)
Nebnext single cell low input cdna synthesis amplification module, by New England Biolabs (1 mentions)
Pacbio sequel, by Pacific Biosciences (1 mentions)
Hiseq x, by Novogene (1 mentions)
Pacbio rs 2 platform, by Pacific Biosciences (1 mentions)
Uniq 10 column trizol total rna extraction kit, by Sangon (1 mentions)
Pacbio sequel system, by Pacific Biosciences (1 mentions)
Hiseq 2500, by Pacific Biosciences (1 mentions)
Hiseq x, by Illumina (1 mentions)
Pacbio rs 2, by Pacific Biosciences (1 mentions)
14 protocols using iso seq
1
Aedes aegypti Genome Annotation
2
Single-Cell Full-Length Transcriptome Sequencing
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IsoSeq SMRTbell libraries were prepared as recommended by the manufacturer (Pacific Biosciences). Briefly, 500 nanograms of total RNA from each sample was used as input for cDNA synthesis using the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB, #E6421L), which employs a modified oligodT primer and template switching technology to reverse-transcribe full-length polyadenylated transcripts. Following double-stranded cDNA amplification and purification, the full-length cDNA was used as input into SMRTbell library preparation, using SMRTbell Express Template Preparation Kit v2.0. Briefly, a minimum of 100 ng of cDNA from each sample was treated with a DNA Damage Repair enzyme mix to repair nicked DNA, followed by an End Repair and A-tailing reaction to repair blunt ends and polyadenylate each template. Next, barcoded overhang SMRTbell adapters were ligated onto each template and purified using 0.6X AMPure PB beads to remove small fragments and excess reagents (Pacific Biosciences). The completed SMRTbell libraries were further treated with the SMRTbell Enzyme Clean Up Kit to remove unligated templates and equimolar pooled together. The final libraries were then annealed to sequencing primer v4 and bound to sequencing polymerase 3.0 before being sequenced on one SMRTcell 8M on the Sequel II system with a 30 h movie.
3
Long-read sequencing of Mus spretus VNO
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Pacific Biosciences Isoseq was used to generate long-read sequences for the VNO from Mus spretus at the Arizona Genome Institute. We sequenced 4 different library sizes 0.8–1.6 kb (× 3 smartcells), 1.3–2.6 kb (× 2 smartcells), 2.2–3.7 kb (× 2 smartcells) and > 3.0 kb (× 2 smartcells) generating a total of 19GB of raw data. These data were run through the PacBio smrtpipe version 2.3 by the Arizona Genome Institute, generating polished high consensus sequences, which we analyzed further for V1R genes.
4
Chromosome-scale Genome Assembly of M. persicae
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To produce a chromosome‐scale genome assembly of the USDA‐Red M. persicae strain, we combined PacBio long‐read, Illumina short‐read and Hi‐C sequencing of genomic DNA. To aid in gene prediction, the M. persicae transcriptome was sequenced using Illumina strand‐specific RNA‐Seq and PacBio Iso‐Seq platforms. Details for the sequencing, assembly and annotation of the M. persicae genome are elaborated in the Supplemental Methods.
5
Herbicide-Sensitive Plant Genome Sequencing
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A single plant from an herbicide-sensitive population (Appels Wilde Samen GmbH, Darmstadt) from Germany was sequenced with CLR in a PacBio Sequel I system. FALCON-Unzip toolkit was used for initial assembly (20 (link)), and contigs were subjected to deduplication with purge_dups v1.0.0 (21 (link)). Hi-C library reads were used as input data for HiRise for chromosome-level scaffolding (22 (link)). To aid gene annotation, both Illumina RNA-seq and PacBio Iso-seq data from five tissues (anthers, whole inflorescences, leaves, pollen, and roots) from the same individual were generated.
6
Transcriptome Analysis of Crab Intestinal Infection
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To identify key genes related to H. eriocheir infection in the E. sinensis intestine, we compared the trimmed high-quality reads from Illumina sequencing against the full-length reference transcriptome which was generated by PacBio Iso-Seq, a new generation of high-throughput sequencing technology. The differential expression of NGS transcripts was analysed by RNA-Seq by Expectation-Maximization (RESM). Transcript and gene read counts were generated from TPM data by correcting for possible gene length variations across samples that were mainly derived from differential transcript usage; for this, we used the tximport 1.10.0 R package and the “length Scaled TPM” tool. Next, the corrected read count data for each gene was used to estimate expression level in terms of fragments per kilobase of transcript per million fragments mapped (FPKM). Then, the corrected read count data for each gene was imported into the EdgeR tool in the R package to identify differentially expressed genes (DEGs) with a fold change ≥ 2.0, a false discovery rate [FDR]-adjusted p-value < 0.05 and an FPKM ≥ 1.
7
Multimodal Sequencing of G. yorkii
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Illumina sequencing of G. yorkii was performed on HiSeq X by Novogene using 550-bp insert DNA libraries prepared at Brigham Young University’s DNA Sequencing Center (BYUDNASC), generating a total of 161.5 Gb of 150-bp paired-end sequencing data. PacBio library preparation and sequencing of HWM G. yorkii DNA was performed on PacBio Sequel by the BYUDNASC. Three SMRT cells were used to generate 198 Gb of continuous long read (CLR) sequencing data.
PacBio Iso-Seq library preparation and sequencing were performed at the BYUDNASC using a single RNA sample created by pooling equal amounts of RNA extracted individually from leaf primordia, mature leaves, shoot apex, mature flowers, flower buds, and roots. One SMRT cell was used to generate 2,341,411 circular consensus sequencing reads (193.3 Mb of data).
PacBio Iso-Seq library preparation and sequencing were performed at the BYUDNASC using a single RNA sample created by pooling equal amounts of RNA extracted individually from leaf primordia, mature leaves, shoot apex, mature flowers, flower buds, and roots. One SMRT cell was used to generate 2,341,411 circular consensus sequencing reads (193.3 Mb of data).
8
Comprehensive Transcriptome Profiling using Iso-Seq
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Total RNA was isolated using a UNIQ-10 column TRIzol total RNA extraction kit (Sangon Biotech) according to the manufacturer’s instructions, followed by treatment with DNase I. The mRNA was purified by a poly T column separation and stored at −80 °C until further analysis. The Iso-Seq library was prepared according to the PacBio Isoform Sequencing protocol (Iso-Seq™). The RNA was reverse transcribed using a SMARTer® PCR cDNA Synthesis Kit and was PCR amplified using KAPA HiFi PCR Kits. These cDNA products were purified using a SMRTbell DNA Template Prep Kit 3.0 for library construction. The libraries were sequenced using P6C4 polymerase and chemistry on a PacBio RS II platform with 240 min movie times at Tianjin Lakeside Powergene Science Development Co. Ltd. In total, 7 SMRT Cells were used to generate 4.4 Gbp of transcriptome cDNA sequencing data.
9
Transcriptome Analysis using PacBio Iso-Seq™
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10
Comprehensive Isoform Profiling of Multiple Primate Species
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Two LCLs per species were used for isoform sequencing experiments (PacBio Iso-Seq; human: GM12878, GM19150; chimpanzee: CH114, CH391; gorilla: DIAN, OMOYE; orangutan: two isogenic cultures of PPY6; macaque: R05040, R02027). In the first run, three libraries of 1–2 kb, 2–3 kb, and 3–6 kb with poly(A) enrichment (Oligo(dT)) were generated using SMRTbell template prep kits and sequenced on a Pacific Biosciences (PacBio) sequel system at Novogene. A total of 26 Gb (subread data) was generated per species, with an average length and N50 of 1980 bp and 2399 bp, respectively. In a second run, poly(A)-enriched libraries (Oligo(dT)) were produced without size selection using SMRTbell template prep kits and sequenced on a PacBio sequel system. In this run, a total of 56 Gb (subread data) was generated per species, with an average length and N50 of 2162 bp and 2647 bp, respectively.
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