The Illumina Hiseq 2500 and PacBio Sequel were both employed to obtain raw data. Initial contigs were assembled from raw NGS data and genome structures were constructed from TGS data. Contig-misassembly was then detected and corrected by CAP3 (75 (link)) and HGAP3 (76 (link)). Tandem repeats were analyzed on RepeatMasker 4.1.1 (77 ) based on Tandem Repeat Finder tool TRF version 4.0.9 (78 (link)) and RepBase (79 (link)).
Hiseq 2500
Manufactured by Pacific Biosciences
The HiSeq 2500 is a high-throughput sequencing system developed by Illumina, a leading provider of DNA sequencing technology. It is designed to generate large volumes of sequence data rapidly and efficiently. The HiSeq 2500 utilizes Illumina's proprietary sequencing-by-synthesis technology to perform DNA sequencing. The core function of the HiSeq 2500 is to enable researchers and scientists to conduct a wide range of genomic and genetic analyses, such as whole-genome sequencing, transcriptome profiling, and targeted resequencing.
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6 protocols using hiseq 2500
1
Hybrid Genome Assembly and Repeat Analysis
2
Mitochondrial DNA Sequencing of Actinidia
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We obtained plant materials of A. arguta, A. chinensis, and A. eriantha from the National Actinidia Germplasm Repository (NAGR; Wuhan, China). For both A. arguta and A. chinensis, intact and pure mitochondria were isolated as previously reported (Tanaka et al. 2004 (link)). The cetyltrimethylammonium bromide method (Doyle 1987 ) was used to extract mtDNA from purified mitochondria. Two different strategies were used for sequencing, in which both a 250-bp paired-end library and 3-kb mate-pair library were prepared and sequenced on an Illumina HiSeq 2500 platform for A. arguta, whereas the PacBio RS II long reads were used for A. chinensis. For A. eriantha, genomic DNA was extracted from fresh leaves, and both Illumina paired-end short reads and PacBio long reads were obtained. The raw Illumina reads were trimmed to exclude low-quality bases and adapter sequences using Trimmomatic v0.36 (Bolger et al. 2014 (link)).
3
Multi-Omics Sequencing of Great Reed Warbler
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High-molecular weight DNA from blood, and total RNA from liver, heart, and muscle tissue, from a juvenile female great reed warbler was extracted and sequenced with the following technologies: PacBio RSII (DNA), Illumina HiSeq X (DNA), chromium linked-read sequencing (10× Genomics; DNA), Bionano (DNA), Illumina HiSeq 2500 (RNA), and PacBio Iso-Seq (RNA). Information on extraction protocols is found in supplementary text 1.1 , Supplementary Material online and details on sequencing technologies in supplementary table 1a , Supplementary Material online .
4
Comparative Genomics of Arthrobacter Isolates
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Whole-genome sequencing of Arthrobacter isolates and their assembly as well as comparative analyses were performed as described previously. Briefly, reads obtained by the Illumina HiSeq 2500 and PacBio Sequell II platforms were assembled and annotated by the A5 and Prokka pipelines, respectively (63 (link), 64 (link)). The AMPHORA and OrthoFinder2 pipelines were used to reconstruct phylogenetic relationships and predict orthologous groups, respectively (40 (link), 41 (link)). Ancestral character estimation was performed by the ace function of the ape R package using the maximum likelihood estimation approach. A full description of methods and algorithms used in this study can be found in Text S1 .
5
Hybrid Assembly of Hymenopteran Genomes
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Genome sequencing was performed using Illumina HiSeq 2500, for paired-end short reads, and PacBio Sequel II platforms. Genome size and heterozygosity were estimated using the GenomeScope 2.0.20 (link),21 (link) The primary assembly was performed using FALCON-UNZIP22 (link) with default settings, and the assembled contigs were polished using Arrow23 (link) and Pilon.24 (link) Completeness of the assembly was assessed using BUSCO v5.2.2 with the ‘insecta_odb10’ and ‘hymenoptera_odb10’ datasets.25 (link) For comparison, we performed BUSCO analysis with other Hymenopteran species: Apis mellifera, Orussus abietinus, Trichogramma pretiosum, Copidosoma floridanum, Nasonia vitripennis, Ceratosolen solmsi, Diachasma alloeum, Fopius arisanus, Microplitis demolitor, Macrocentrus cingulum, Cotesia typhae, Cotesia glomerata, Chelonus insularis, and Aphidius gifuensis. The references of the genomes of all species are listed in Supplementary Fig. S1 .26–38 (link)
6
Comprehensive Genome Characterization of Strain L14
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Genomic DNA of strain L14 was extracted with Gentra Puregene Yeast/Bact. Kit (Qiagen, Valencia, CA, USA) officially recommended by Nanopore [13 (link)]. The next-generation and third-generation sequencing technologies were, respectively, utilized Illumina HiSeq 2500 platform and PacBio Sequel platform. DNA libraries of a certain concentration and volume were added to each individual flow cell and the flow cells were transferred to a Grid ION X5 sequencer (Nanopore, Oxford, UK) for real-time single-molecule sequencing. Through a variety of methods, including Homology, SNAP, Augustus, coding genes, repetitive sequences, non-coding RNA, etc., were predicted to obtain the composition of the sequenced strain genome. Repeat sequence prediction was performed by Proteinmask, repeatmasker and trf. The tRNA region was predicted by tRNAscan software, rRNA was predicted by RNAmmer software, miRNA and snRNA were achieved by comparison with Rfam database.
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