Solid5500

Manufactured by Thermo Fisher Scientific

Sourced in United States

The SOLiD5500 is a next-generation sequencing (NGS) platform designed for high-throughput DNA sequencing. It utilizes sequencing-by-ligation technology to generate large volumes of high-quality sequence data. The core function of the SOLiD5500 is to perform DNA sequencing, providing users with the ability to analyze genetic information.

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6 protocols using solid5500

Shotgun Metagenome Sequencing Protocols

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The “shotgun” libraries were prepared for the sequencing platforms SOLiD 4, SOLiD 5500 and Ion Torrent (Life Technologies, USA). The sequencing was performed according to the manufacturer’s instructions. For SOLiD 4, the SOLiD Fragment Library Construction Kit, SOLiD Fragment Library Barcoding Module 1–16, SOLiD EZ Bead TM E80 System Consumables, SOLiD ToP Sequencing Kit and MM50/5 (Life Technologies, USA) were used. For SOLiD 5500, the 5500 SOLiD Fragment Library Core Kit, SOLiD Fragment Library Barcoding Kit, SOLiD FlowChip Kit, SOLiD FWD SR S50 Kit and SOLiD Run Cycle Buffer Kit (Life Technologies, USA) were used. For Ion Torrent PGM, the Ion Xpress Plus Fragment Library Kit, Ion Sequencing Kit, Ion PGM Template OT2 200 Kit, Ion PGM Sequencing 200 Kit and Ion 318 Chip Kit (Life Technologies, USA) were used.
Three of the samples were sequenced using more than one platform; in total, 28 metagenomic read sets were obtained (Additional file 1: Table S8).

Tyakht A.V., Manolov A.I., Kanygina A.V., Ischenko D.S., Kovarsky B.A., Popenko A.S., Pavlenko A.V., Elizarova A.V., Rakitina D.V., Baikova J.P., Ladygina V.G., Kostryukova E.S., Karpova I.Y., Semashko T.A., Larin A.K., Grigoryeva T.V., Sinyagina M.N., Malanin S.Y., Shcherbakov P.L., Kharitonova A.Y., Khalif I.L., Shapina M.V., Maev I.V., Andreev D.N., Belousova E.A., Buzunova Y.M., Alexeev D.G, & Govorun V.M. (2018). Genetic diversity of Escherichia coli in gut microbiota of patients with Crohn’s disease discovered using metagenomic and genomic analyses. BMC Genomics, 19, 968.

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Trio Whole Exome Sequencing for Variant Discovery

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Genomic DNA from the subject and his parents was isolated from peripheral blood using a standard phenol chloroform protocol. DNA purification was performed using Amicon Ultra 30K centrifugal filter units (Millipore). Bar-coded DNA libraries were prepared and exome capture (Agilent 50 Mb SureSelect Human All Exon V3) and sequencing (Life Technologies SOLiD5500) was performed for the trio. Resulting reads (75 bp) were generated with Exact Call Chemistry and aligned to the human reference sequence (hg19) with LifeScope (Life Technologies), and variants were called with GATK and annotated with in-house software (PolyWeb). Mean coverage was 76–92x, with 86− 87% of bases covered >15x. Variants present in databases (dbSNP, 1000 Genomes, Exome Sequencing Project) or previously observed in 1029 in-house exomes were excluded and de novo coding or splicing variants predicted to be damaging by SIFT (Kumar et al., 2009 (link)) or PolyPhen-2 (Adzhubei et al., 2013 ) were verified via Sanger sequencing with standard protocols (Applied Biosystems 3130xl). Additional prediction tools LRT (Chun and Fay, 2009 (link)) and MutationTaster (Schwarz et al., 2010 (link)) were used.

Duchatelet S., Boyden L.M., Ishida-Yamamoto A., Zhou J., Guibbal L., Hu R., Lim Y.H., Bole-Feysot C., Nitschké P., Santos-Simarro F., de Lucas R., Milstone L.M., Gildenstern V., Helfrich Y.R., Attardi L.D., Lifton R.P., Choate K.A, & Hovnanian A. (2018). Mutations in PERP cause dominant and recessive keratoderma. The Journal of investigative dermatology, 139(2), 380-390.

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Transcriptomic Analysis of Crb1/Crb2 Knockout Mice

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Sequencing was performed using Life Technologies SOLiD5500 with single-end, 50-bp reads. Two separate runs were performed. Run 1 contained 30 mice; n = 5 Crb1^KOCrb2^F/F (Crb1^KO) and n = 5 Crb1^KOCrb2^ΔRPC (Tg) per development time point of the mouse embryo at embryonic day 15.5 (E15.5), E17.5, and P1. Run 2 contained 9 mice; n = 4 Crb1^KOCrb2^ΔRPC mice (C57BL/6JOlaHsd) and n = 5 healthy mice (C57BL/6JRccHsd = wild type [WT]) at E15.5. Reads were aligned against mm10 using the “whole.transcriptome.frag” workflow with the bamgen.mqv.threshold set to 20 (Lifescope v.2.5). Counts were obtained using the gene transfer format (GTF) output format as supplied by Lifescope (transformation of refGene.txt downloaded from the University of California Santa Cruz [UCSC] Table Browser on June 25, 2014) within this workflow.

Buck T.M., Quinn P.M., Pellissier L.P., Mulder A.A., Jongejan A., Lu X., Boon N., Koot D., Almushattat H., Arendzen C.H., Vos R.M., Bradley E.J., Freund C., Mikkers H.M., Boon C.J., Moerland P.D., Baas F., Koster A.J., Neefjes J., Berlin I., Jost C.R, & Wijnholds J. (2023). CRB1 is required for recycling by RAB11A+ vesicles in human retinal organoids. Stem Cell Reports, 18(9), 1793-1810.

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Genetic Profiling of Ciliopathies in Clinical Cases

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All patients consented to this study. Ethical approval was obtained from the National Research Ethics Service (NRES) Committee North East (14/NE/1076). Following informed and written consent, urine and blood samples were obtained from an affected 4 year-old boy with clinical JBTS with a retinal, renal, and cerebellar phenotype, a 12 year old boy with isolated LCA and his unaffected sibling and healthy gender and age-matched controls. Ethical approval was obtained from the National Research Ethics Service (NRES) Committee North East (14/NE/1076). All methods were performed in accordance with the relevant ethical guidelines and regulations.
Sequencing of ciliopathy genes was performed by applying exon-enriched NGS targeting up to 1, 221 genes associated with cilia including all known genes associated with ciliopathies (“ciliome sequencing”) (44–46 (link)) and consecutive barcoded NGS on a SoliD5500 (Life tech) or HiSeq 2500 (Illumina) platform. Confirmation of mutations and segregation analysis was performed on other family members following informed consent using Sanger sequencing, using exon specific primers.

Srivastava S., Ramsbottom S.A., Molinari E., Alkanderi S., Filby A., White K., Henry C., Saunier S., Miles C.G, & Sayer J.A. (2017). A human patient-derived cellular model of Joubert syndrome reveals ciliary defects which can be rescued with targeted therapies. Human Molecular Genetics, 26(23), 4657-4667.

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Whole Exome Sequencing of Multiple Cancers

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Serial samples of normal tissue, atypical adenomatous hyperplasia (AAH), and MPM tissue from the patient with multiple cancers including MPM were used for the exome sequencing library preparation. SOLiD 5500 (Life Technologies) was used for whole exome sequencing according to the manufacturer's protocol and our previous experiment [32 (link)]. Briefly, 3 ug of DNA was fragmented by Covaris S220 (Covaris), and the fragmented DNA was barcode ligated and amplified. After fragmented DNA was quantified, 500 ng of DNA was used for the TargetSeq Exome Enrichment kit (Life Technologies) to enrich the exome only. A diluted exome library was amplified by emulsion PCR and enriched. The prepared libraries were run onto the Flow Chip in SOLiD 5500.

Kang H.C., Kim H.K., Lee S., Mendez P., Kim J.W., Woodard G., Yoon J.H., Jen K.Y., Fang L.T., Jones K., Jablons D.M, & Kim I.J. (2016). Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas. Oncotarget, 7(7), 8321-8331.

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Exome Sequencing with Agilent SureSelect

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Agilent SureSelect libraries were prepared from 3 μg of genomic DNA sheared with a Covaris S2 Ultrasonicator as recommended by the manufacturer. Exome capture was performed with the 50 Mb SureSelect Human All Exon kit (Agilent Technologies) using a multiplex approach with molecular barcodes for traceable identification of samples. Sequencing was carried with the SOLiD5500 (Life Technologies) on a pool of barcoded exome libraries. 75 + 35 paired-end reads were generated and mapped on the human genome reference (NCBI build37/hg19 version) using LifeScope (Life Technologies).
For each subject, sequences produced allowed a mean sequence coverage between 42–87 reads per bp. The average coverage was 70×, with more than 75% of targeted bases covered 15×. Sequence reads were aligned to the human reference genome sequence (assembly GRCh37) using Mapreads. SNPs and indels were called using Genome Analysis Toolkit and Picard Tools. Poorly mapped (less than 3× cover) and low-quality reads (quality score less than 20) were removed. In-house software (PolyWeb) was used to annotate and filter the variants.

Mircsof D., Langouët M., Rio M., Moutton S., Siquier-Pernet K., Bole-Feysot C., Cagnard N., Nitschke P., Gaspar L., Žnidarič M., Alibeu O., Fritz A.K., Wolfer D.P., Schröter A., Bosshard G., Rudin M., Koester C., Crestani F., Seebeck P., Boddaert N., Prescott K., Hines R., Moss S.J., Fritschy J.M., Munnich A., Amiel J., Brown S.A., Tyagarajan S.K, & Colleaux L. (2015). Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects. Nature neuroscience, 18(12), 1731-1736.

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