Cbot 2 system

Manufactured by Illumina

The CBot 2 system is a laboratory instrument designed for automated cluster generation for Illumina sequencing platforms. It prepares DNA libraries for sequencing by performing the critical steps of bridge amplification, which generates clusters of clonal DNA fragments on the flow cell surface. The CBot 2 system provides a streamlined and automated workflow to produce high-quality cluster-amplified DNA samples ready for sequencing.

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7 protocols using cbot 2 system

RNA Sequencing Library Preparation and Sequencing

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Total RNA was prepared with the RNeasy Plus kit (Qiagen, Germantown, MD). For RNA sequencing, RNA purity and integrity was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and an Agilent Bioanalyzer 2100 with Agilent RNA Nano 6000 chips, respectively. Sequencing of RNA was performed as described in (Szwarc et al. 2018 (link)). Briefly, libraries were prepared using TruSeq RNA library preparation kit v2 (Illumina, Inc., San Diego, CA) and enriched by PCR. Adapter-ligated fragment concentration was measured by quantitative PCR assay with a KAPA library Quant kit (Kapabiosystems, Inc., Wilmington, MA). Samples were then pooled and quantified again by qPCR. Bridge amplification using the cBot 2 system (Illumina, Inc.) was used for clonal cluster generation of library pools. Indexed paired-end sequencing was performed with the HiSeq 2500 sequencing system (Illumina, Inc.).

Szwarc M.M., Hai L., Gibbons W.E., White L.D., Mo Q., Kommagani R., Lanz R.B., DeMayo F.J., O’Malley B.W, & Lydon J.P. (2018). Retinoid Signaling Controlled by SRC-2 in Decidualization Revealed by Transcriptomics. Reproduction (Cambridge, England), 156(5), 387-395.

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Whole-Genome Sequencing of PARK2 Carriers

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To dissect the novel GWAS signal associated with AAO identified in the Spanish population, we performed whole-genome sequencing (WGS) analyses in 5 of 37 homozygous carriers of the PARK2 signal. DNA concentration was determined by Qubit fluorescence and normalized to 20 ng/uL. One microgram of total genomic DNA was sheared to a target size of 450 base pairs (bp) using the Covaris LE220 ultrasonicator. Library preparation was achieved using the TruSeq DNA PCR-Free High Throughput Library Prep Kit and IDT for Illumina TruSeq DNA UD Indexes (96 Indexes, 96 Samples). Sequencing libraries were assessed for size distribution, absence of free adapters, and adapter dimers on a Fragment Analyzer. Library quantitation was performed by quantitative polymerase chain reaction using the KAPA Library Quantification Kit subsequent normalization to 4 nM. Libraries were clustered on v2.5 flowcell using the Illumina cBot 2 System before sequencing on the Illumina HiSeq X System using paired-end 150-bp reads. BCL files processed with alignment by ISAAC on HAS 2.2 and BAMs were used for QC assessment of mean coverage, percent duplicates, percent bases >20× coverage, and percent noise sites.

Bandres-Ciga S., Ahmed S., Sabir M.S., Blauwendraat C., Adarmes-Gómez A.D., Bernal-Bernal I., Bonilla-Toribio M., Buiza-Rueda D., Carrillo F., Carrión-Claro M., Gómez-Garre P., Jesús S., Labrador-Espinosa M.A., Macias D., Méndez-del-Barrio C., Periñán-Tocino T., Tejera-Parrado C., Vargas-González L., Diez-Fairen M., Alvarez I., Tartari J.P., Buongiorno M., Aguilar M., Gorostidi A., Bergareche J.A., Mondragon E., Vinagre-Aragon A., Croitoru I., Ruiz-Martínez J., Dols-Icardo O., Kulisevsky J., Marín-Lahoz J., Pagonabarraga J., Pascual-Sedano B., Ezquerra M., Cámara A., Compta Y., Fernández M., Fernández-Santiago R., Muñoz E., Tolosa E., Valldeoriola F., Gonzalez-Aramburu I., Sanchez Rodriguez A., Sierra M., Menéndez-González M., Blazquez M., Garcia C., Suarez-San Martin E., García-Ruiz P., Martínez-Castrillo J.C., Vela-Desojo L., Ruz C., Barrero F.J., Escamilla-Sevilla F., Mínguez-Castellanos A., Cerdan D., Tabernero C., Gomez Heredia M.J., Perez Errazquin F., Romero-Acebal M., Feliz C., Lopez-Sendon J.L., Mata M., Martínez Torres I., Kim J.J., Dalgard C.L., Brooks J., Saez-Atienzar S., Gibbs J.R., Jorda R., Botia J.A., Bonet-Ponce L., Morrison K.E., Clarke C., Tan M., Morris H., Edsall C., Hernandez D., Simon-Sanchez J., Nalls M.A., Scholz S.W., Jimenez-Escrig A., Duarte J., Vives F., Duran R., Hoenicka J., Alvarez V., Infante J., Marti M.J., Clarimón J., López de Munain A., Pastor P., Mir P, & Singleton A. (2019). The Genetic Architecture of Parkinson Disease in Spain: Characterizing Population-Specific Risk, Differential Haplotype Structures, and Providing Etiologic Insight. Movement disorders : official journal of the Movement Disorder Society, 34(12), 1851-1863.

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RNA-Seq Transcriptome Analysis Protocol

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Samples from three independent experiments were used for RNA sequencing. For each sample, an indexed cDNA library was prepared from 1 μg total RNA using the KAPA‐stranded mRNA‐seq kit (Sopachem, Ochten, the Netherlands). Clusters were generated using the Cbot2 system (Illumina, San Diego, California), and amplified cDNA fragments were sequenced on a HiSeq 4000 system (Illumina) as follows: 51 cycles for read 1 and 8 cycles for index 1. The raw sequenced reads were mapped to the human reference genome build GRCh38 using spliced transcripts alignment to a reference (STAR).³⁷ Mapped reads were quantified using RSEM³⁸ for accurate quantitation resulting in, on average, 34 740 890 ± 8 771 147 counts per sample. After autoscaling, the resulting data were first summarized by principal component analysis (PCA) using the flashPCA (R package). Plotly was used to generate interactive graphs (2D plots). Heatmap analysis was performed using the heatmaply package. RNA‐sequencing data are available in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/experiments/E‐MTAB‐8392/) under accession E‐MTAB‐8392.

Tiemeier G.L., de Koning R., Wang G., Kostidis S., Rietjens R.G., Sol W.M., Dumas S.J., Giera M., van den Berg C.W., Eikenboom J.C., van den Berg B.M., Carmeliet P, & Rabelink T.J. (2020). Lowering the increased intracellular pH of human‐induced pluripotent stem cell‐derived endothelial cells induces formation of mature Weibel‐Palade bodies. Stem Cells Translational Medicine, 9(7), 758-772.

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Comprehensive Genomic Profiling Protocol

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DNA sequencing was performed on the Illumina HiSeq X 2 × 150 bp run (Illumina). The final DNA library was diluted, denatured, and introduced into the lanes of the flow cell using the Illumina cBot 2 system according to the manufacturer's protocol. The libraries were loaded at a 2:1 tumor:normal ratio to reach coverage (average read depth) of 80× for the tumor sample and 40× for the normal sample. RNA libraries were sequenced on the Illumina HiSeq 2500 2 × 50 bp Rapid Run platform multiplexing a total of seven samples onto one flow cell giving a minimum of 40,000 reads per sample.

Diolaiti D., Dela Cruz F.S., Gundem G., Bouvier N., Boulad M., Zhang Y., Chou A.J., Dunkel I.J., Sanghvi R., Shah M., Geiger H., Rahman S., Felice V., Wrzeszczynski K.O., Darnell R.B., Antonescu C.R., French C.A., Papaemmanuil E., Kung A.L, & Shukla N. (2018). A recurrent novel MGA–NUTM1 fusion identifies a new subtype of high-grade spindle cell sarcoma. Cold Spring Harbor Molecular Case Studies, 4(6), a003194.

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RNA-seq Library Preparation and Sequencing

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The integrity and concentration of the extracted RNA was assessed on the LabChip GXII Touch HT using the RNA Assay and the DNA 5K/RNA/Charge Variant Assay LabChip (PerkinElmer). Sequencing mRNA libraries were constructed from 500 ng of total RNA using the Illumina TruSeqStranded mRNA kit (Illumina). cDNA was synthesized using Superscript IV Reverse Transcriptase (Thermo Fisher) and amplified for 15 cycles after ligating with TruSeq RNA Combinatorial Dual-Index adapters. Clonal amplification and cluster generation was performed using Illumina’s cBot 2 System. Sequencing libraries were run on Illumina HiSeq platforms using 75 bp (93% of samples) or 150 bp (7% of samples) paired-end reads at the Clinical Genomics Laboratory, Sidra Medicine. We targeted a coverage of 20 M reads per sample. Obtained coverage was 18.4 M (s.d. 4.7 M).

Roelands J., Kuppen P.J., Ahmed E.I., Mall R., Masoodi T., Singh P., Monaco G., Raynaud C., de Miranda N.F., Ferraro L., Carneiro-Lobo T.C., Syed N., Rawat A., Awad A., Decock J., Mifsud W., Miller L.D., Sherif S., Mohamed M.G., Rinchai D., Van den Eynde M., Sayaman R.W., Ziv E., Bertucci F., Petkar M.A., Lorenz S., Mathew L.S., Wang K., Murugesan S., Chaussabel D., Vahrmeijer A.L., Wang E., Ceccarelli A., Fakhro K.A., Zoppoli G., Ballestrero A., Tollenaar R.A., Marincola F.M., Galon J., Khodor S.A., Ceccarelli M., Hendrickx W, & Bedognetti D. (2023). An integrated tumor, immune and microbiome atlas of colon cancer. Nature Medicine, 29(5), 1273-1286.

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Illumina DNA and RNA Sequencing Protocol

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DNA sequencing is performed on the Illumina HiSeq X 2 × 150 bp run (Illumina, San Diego, CA). The final DNA library was diluted, denatured, and introduced into the lanes of the flow cell by using the Illumina cBot 2 system according to the manufacturer's protocol. The libraries were loaded at a 2:1 tumor/normal (T/N) ratio to reach coverage (average read depth) of 80× for the tumor sample and 40× for the normal sample. A total of two patient samples, or T/N pairs, can be loaded onto one HiSeqX flowcell. For RNA samples prepared with the mRNA protocol, libraries were sequenced on the Illumina HiSeq 2500 2 × 125 bp Rapid Run platform. For RNA samples prepared with the KAPA total protocol, libraries were sequenced on the Illumina HiSeq 2500 2 × 50 bp Rapid Run platform. A total of seven samples were multiplexed onto one flowcell, giving a minimum of 40 million reads per sample.

Wrzeszczynski K.O., Felice V., Abhyankar A., Kozon L., Geiger H., Manaa D., London F., Robinson D., Fang X., Lin D., Lamendola-Essel M.F., Khaira D., Dikoglu E., Emde A.K., Robine N., Shah M., Arora K., Basturk O., Bhanot U., Kentsis A., Mansukhani M.M., Bhagat G, & Jobanputra V. (2018). Analytical Validation of Clinical Whole-Genome and Transcriptome Sequencing of Patient-Derived Tumors for Reporting Targetable Variants in Cancer. The Journal of Molecular Diagnostics : JMD, 20(6), 822-835.

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RNA-seq Library Preparation and Sequencing

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Total RNA was evaluated for its quantity and quality using an Agilent Bioanalyzer 2100; RNA integrity numbers were all greater than 9. Fifty nanograms of total RNA was used for each sequencing library. cDNA library preparation was carried out at the Center for Medical Genomics, and included mRNA enrichment using oligo dT-beads to capture polyA RNA, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the KAPA mRNA Hyper Prep Kit Technical Data Sheet, KR1352 – v4.17 (Roche Corporate). Each resulting indexed library was quantified and its quality accessed by Qubit and Agilent Bioanalyzer, and all libraries were pooled at equal molarity. Five microliter aliquots of 2 nM pooled libraries per lane were denatured, neutralized and applied to flow cell deposition on both surfaces of the flow cell; library cluster amplification was performed on a cBot 2 System (Illumina, Inc.). The resulting flow cell was sequenced on an Illumina HiSeq 4000, 75 base paired-end sequencing (Illumina, Inc.). Approximately 30 M reads per library were generated (minimum 26.5 M, maximum 35.4 M). A Phred quality score (Q score) was used to measure the quality of sequencing. More than 90% of the sequencing reads reached Q30 (99.9% base call accuracy).

McClintick J.N., Thapa K., Liu Y., Xuei X, & Edenberg H.J. (2020). Effects of chronic intermittent ethanol exposure and withdrawal on neuroblastoma cell transcriptome. Alcohol (Fayetteville, N.Y.), 85, 119-126.

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