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Whole Genome Sequencing (WGS) is a robust methodology utilized in genomics research, personalized medicine, and clinical diagnostics. It entails determining the complete DNA sequence of an organism’s genome, providing understanding of genetic makeup and insights into genetic variations, evolutionary patterns, and disease mechanisms. WGS comprises: DNA extraction, library preparation involving fragmentation of DNA and addition of adapters, and sequencing using next-generation sequencing (NGS) platforms.

Bioinformatic analysis of WGS data involves assembling sequencing reads to reconstruct the genome sequence, annotating the genome, and calling variants to pinpoint differences between the sequenced genome and a reference. Illumina DNA PCR-Free library preparation protocol combines on-bead tagmentation and PCR-free chemistry. This approach ensures uniform coverage across the genome and is suitable for human WGS, de novo assembly of microbial genomes, and tumor–normal variant calling.

Whole Exome Sequencing (WES) is a targeted approach focusing on the protein-coding regions of the genome (around 1-2%). The exome encompasses a significant portion of known disease-causing mutations, it is an affordable choice for various genetic studies where focusing on protein-coding regions suffices for research or clinical objectives.

WES begins with DNA extraction, followed by library preparation involving fragmenting DNA and adding adapters. Next is exome capture, where target-specific probes enrich DNA fragments corresponding to exonic regions, the prepared exome library undergoes high-throughput sequencing.

Bioinformatic analysis includes aligning reads to a reference genome and identifying genetic variants within exonic regions, such as single nucleotide variants (SNVs), insertions, deletions, and structural variations. In the NF Genomics protocol, TWIST Comprehensive Exome Panel is utilized, covering over 99% of protein-coding genes, with a design size of 41.2 Mb and targeting a total of 36.8 Mb, including an expanded content of RefSeq and GENCODE databases.

The Visium Spatial Gene Expression solution by 10X Genomics enables spatial profiling of gene expression in intact tissue sections, preserving cellular spatial context. For fresh-frozen tissues, the protocol involves sectioning, mounting, fixation, permeabilization, reverse transcription, library preparation, sequencing, and spatial mapping. For FFPE tissues, additional steps include deparaffinization, rehydration, antigen retrieval, and tissue mounting with Visium Cytassist. Both protocols end with library preparation, sequencing, data analysis, and spatial mapping to retain spatial information.

Total RNA sequencing comprehensively analyzes the transcriptome, revealing mRNA and non-coding RNA profiles. This technique aids in understanding gene expression, identifying novel transcripts, and deciphering regulatory mechanisms in various biological processes. The process involves RNA extraction, ribosomal RNA depletion, cDNA library generation via reverse transcription, fragmentation, adaptor ligation, PCR amplification, high-throughput sequencing, and bioinformatic analysis for gene expression quantification and transcript discovery.

Libraries are prepared using the Illumina Total RNA Prep protocol (Illumina) with Ribo-Zero Plus that supports diverse RNA inputs, including FFPE and low-quality samples, and incorporates Ribo-Zero Plus or Ribo-Zero Plus Microbiome for efficient removal of abundant RNA from various species, including human, mouse, rat, bacteria, and complex microbial samples like stool, ideal for meta-transcriptomic studies.

Small RNA sequencing is crucial for studying short RNA molecules (18-30 nt) involved in gene regulation. It analyzes miRNAs and small RNAs, shedding light on gene regulation, development, and disease. The process involves RNA extraction, size selection, adapter ligation, library amplification, size selection, sequencing, and bioinformatics analysis to identify and quantify small RNAs. High-throughput sequencing reveals small RNA profiles, which are analyzed through bioinformatics to identify miRNAs, siRNAs, and piRNAs, aiding in understanding their roles in cellular processes.

Libraries are prepared using the SMARTer smRNA-Seq Kit (Takara) that is compatible with total RNA or enriched small RNA, incorporating SMART technology and primers with locked nucleic acids (LNAs). Users can analyze various small RNA species and create complex sequencing libraries. Illumina adapters and index sequences are added during library amplification, ensuring unbiased representation of diverse small RNA species.

Single-cell multiome ATAC + Gene Expression technology by 10X Genomics enables concurrent profiling of chromatin accessibility and gene expression at the single-cell level, offering a holistic view of cellular molecular landscapes. It aids in understanding cellular diversity, identifying cell types, deciphering regulatory networks, and correlating chromatin accessibility with gene expression.

The ATAC-seq component assesses the accessibility of chromatin, offering insights into regions of the genome that are open and accessible for transcription factors and other regulatory elements. Concurrently, the RNA-seq component captures messenger RNA transcripts present in each nucleus, shedding light on active genes and their expression levels.

The workflow involves cell isolation, nuclei preparation, transposase reaction for ATAC-seq, GEM formation, reverse transcription for RNA-seq, library preparation, high-throughput sequencing, and specialized bioinformatics analysis for data processing.

Single-cell immune profiling with 10x Genomics technology enables 5′ RNA sequencing at the single-cell level, alongside profiling T-cell and/or B-cell receptors at single-cell resolution by sequencing V(D)J regions. Cells are isolated into droplets, where RNA is barcoded at the 5′ end. cDNA synthesis and targeted amplification yields three types of libraries: GEX libraries for single-cell gene expression analysis, and libraries for TCR and BCR gene profiling. Pooled libraries undergo high-throughput Illumina sequencing. Bioinformatics tools demultiplex reads, assign to cells based on barcodes, and identify T and B cell clones, providing insights into gene expression and immune cell repertoire diversity and clonal expansion.

Single-cell ATAC sequencing with 10X Genomics profiles chromatin accessibility of individual cells/nuclei, revealing cellular heterogeneity and regulatory processes. The scATAC-seq protocol involves cell isolation, nuclei extraction, transposase reaction for chromatin fragmentation and adapter addition, GEM formation for nuclei encapsulation with unique barcodes, library preparation via PCR amplification, high-throughput sequencing, and data analysis to identify accessible chromatin regions.

Single-cell 3′ RNA sequencing with 10x Genomics technology allows gene expression study at a single-cell level, profiling thousands to tens of thousands of cells in parallel. It captures cellular heterogeneity, identifies rare cell types, and discerns gene expression differences, enhancing understanding of cellular diversity. The process involves cell isolation into droplets, RNA capture and barcoding and cDNA synthesis, library preparation, pooling, sequencing, and bioinformatic analysis for single-cell transcriptomic profiling.

The Single Cell Gene Expression Flex assay by 10X Genomics, enables single-cell/nuclei RNA-seq libraries from formaldehyde-fixed cells and tissues, including FFPE blocks. Utilizing sequence-specific probe pairs for about 18,000 human and 19,000 mouse genes, this method accounts for RNA degradation. Sample multiplexing, facilitated by sample barcodes in probes, allows pooling of discrete cell populations, accommodating up to 16 samples and potentially analyzing 128,000 cells per GEM reaction.

The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the NovaSeq6000 sequencing platform (Illumina).

The NovaSeq 6000 sequencing platform by Illumina offers high-throughput capabilities, making it ideal for diverse genomic studies. With its scalability and sequencing-by-synthesis technology, it efficiently generates large amounts of sequencing data. Its dual-flow cell design allows parallel sequencing of multiple samples, reducing turnaround times. Common applications include whole genome sequencing, exome sequencing, transcriptome analysis (bulk and single-cell RNA-Seq), metagenomics, epigenomics, and population genomics. This platform is pivotal for comprehensive genomic research, from understanding genetic variations to characterizing microbial communities and studying epigenetic modifications.

The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the NextSeq2000 sequencing platform (Illumina).

The NextSeq 2000, an Illumina platform, is renowned for its high-throughput benchtop sequencing capabilities, offering flexibility and scalability for diverse applications. It employs sequencing-by-synthesis technology, capturing emitted fluorescence to determine DNA sequences. With versatile configurations, it supports various flow cell setups and sequencing kits, catering to different project scales. Commonly used for whole genome sequencing (WGS), RNA sequencing (RNA-Seq), targeted sequencing (including amplicon sequencing and target capture), exome sequencing, metagenomics, and ChIP-Seq and epigenomics studies, the NextSeq 2000 is pivotal in genomics research, enabling comprehensive analysis of genetic and epigenetic features across diverse biological samples.

The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the MiSeq sequencing platform (Illumina).

The MiSeq, an Illumina platform, is a compact benchtop sequencer ideal for smaller-scale sequencing projects. It employs sequencing-by-synthesis technology, enabling the determination of DNA sequences via fluorescently labeled nucleotides. With compatibility with various reagent kits and chemistries, users can tailor sequencing protocols to their experiment needs. Commonly used for targeted sequencing (amplicon sequencing, target capture, and custom panels), small genome sequencing (bacterial or viral), metagenomics, 16S rRNA sequencing, small RNA-Seq for gene expression profiling, and viral genome sequencing, the MiSeq facilitates diverse applications in genomics research, particularly in microbial diversity studies and viral evolution analysis.

The native barcoding kit enhances nanopore sequencing by enabling simultaneous sequencing of multiple samples with unique barcodes. After gDNA extraction, each sample is tagged with a barcode during library preparation. Sequencing generates long reads, allowing real-time base calling. Bioinformatics tools can be used to analyze data, including aligning reads and variant calling. This approach is particularly useful for studying small bacteria genomes because it is cost-effective and efficient, beneficial for microbiome studies and environmental monitoring and other applications in microbial genomics.

Libraries are prepared with the Native Barcoding Kit 96 V14 that enables PCR-free multiplexing of small gDNA samples using 96 unique barcodes. It involves repairing and dA-tailing gDNA, then ligating a unique dT-tailed barcode adapter. Barcoded samples are pooled, and each barcode adapter ligates to a sequencing adapter. Optimized for high sequencing accuracies (>99% Q20+) on nanopore Flowcells R10.4.1.

Nanopore sequencing directly sequences DNA using nanopores, known for producing long or ultra-long reads. Protocol includes DNA extraction, minimal fragmentation for long reads or no fragmentation for ultra-long reads generation, DNA end repair, adapter ligation, device setup, sequencing, and data collection through electrical signals. Base-calling converts signals into DNA sequence. Data analysis involves error correction, read filtering, and read assembly.

The library prep method for long-read sequencing involves repairing DNA ends, dA-tailing, and ligating sequencing adapters. It achieves >99% sequencing accuracy (Q20+) on R10.4.1 flow cells. It is compatible with target enrichment, whole genome amplification, and size selection.

The Ultra-Long DNA Sequencing library prep method facilitates preparation of ultra-high molecular weight DNA, yielding N50s >50 kb and reads up to 4+ Mb. Utilizing transposase chemistry, it cleaves template molecules and attaches tags, followed by rapid adapter addition.

Direct RNA Sequencing with Nanopore technology sequences RNA molecules without cDNA conversion, offering real-time detection of RNA sequences and modifications. It preserves RNA’s native state, revealing insights into RNA processing and modifications. RNA extraction, adapter ligation, sequencing, and data analysis are key steps. Raw signals are base-called for RNA sequence reconstruction. This method can offer a comprehensive view of the transcriptome.

The Direct RNA Sequencing Kit (SQK-RNA004) facilitates native RNA sequencing, avoiding cDNA conversion. It supports poly(A)-tailed RNA or total RNA like eukaryotic mRNA and viral RNA. This upgrade enhances sequencing output and accuracy on the latest RNA flow cells (FLO-MIN004RA and FLO-PRO004RA). It includes reformulated priming reagents for flow cell compatibility and features fuel fix technology for extended experiment runs without additional fuel.

Cell-free DNA (cfDNA) sequencing with Nanopore technology allows direct interrogation of genetic information in circulating DNA without PCR amplification. This real-time protocol enables methylation status analysis. It offers insights into cfDNA’s genomic landscape, benefiting clinical diagnostics like cancer detection, treatment monitoring, and minimal residual disease identification. The protocol involves cfDNA extraction, library preparation, loading onto a Nanopore sequencing device, real-time sequencing, and data analysis for variant calling, copy number and methylation status analysis.

The library preparation involves repairing DNA ends, dA-tailing, and ligating sequencing adapters. The kit ensures high sequencing accuracies (Q20+) on nanopore Flowcells R10.4.1, with updates for enhanced DNA capture and fuel fix technology for longer runs. The protocol is optimized for short DNA fragments recovery, based on a modified long-reads protocol.

Nanopore cDNA sequencing allows the exploration of gene expression dynamics, alternative splicing, and RNA biology with long-read capabilities. The process involves RNA extraction, cDNA synthesis, library preparation with unique barcodes, loading onto a nanopore sequencer, real-time sequencing of cDNA strands, base calling, and subsequent bioinformatics analysis for aligning reads, identifying gene isoforms, and quantifying gene expression.

The libraries are prepared with the PCR-cDNA Sequencing Kit that enables nanopore sequencing of cDNA from low input poly(A)+ RNA or total RNA with additional optimization. It employs a strand-switching method to select full-length transcripts and incorporates unique molecular identifiers (UMIs). The kit includes the Rapid Adapter T (RAP T) for enhanced capture and fuel fix technology for longer experiments without fuel addition. A new cDNA RT adapter and RT primer reduce overlaps during reverse transcription and allow measurement of polyA+ tail lengths.

mRNA sequencing analyzes the transcriptome, revealing gene expression patterns and novel transcripts in cells or tissues. The process involves RNA isolation, cDNA synthesis via reverse transcription, library preparation with added adapters, high-throughput sequencing, and bioinformatic analysis mapping reads to a reference genome or transcriptome to discern gene expression levels and novel transcripts.

mRNA sequencing from standard RNA input is performed with Illumina Stranded mRNA Prep protocol (Illumina) that ensures precise strand orientation measurement, uniform coverage, and high-confidence detection of novel features like isoforms and gene fusions.

mRNA sequencing from RNA low input is performed with the SMART-Seq v4 PLUS Kit (Takara). SMART technology ensures full-length transcript information, enabling analysis of isoforms, gene fusions, and mutations, with improved gene detection via locked nucleic acid (LNA) technology. High reproducibility and accurate coverage of GC-rich transcripts are ensured.

Microbiome analysis via 16S and ITS amplicon sequencing is pivotal for studying microbial community composition and diversity, spanning bacteria and fungi. The 16S rRNA gene, found in bacteria and archaea, and ITS for fungi are key markers used for taxonomic classification.

The process entails sample collection, DNA extraction, PCR amplification targeting variable regions of 16S rRNA or ITS genes, library preparation, and high-throughput sequencing. Taxonomic classification involves clustering reads into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs), followed by comparison to reference databases. Diversity and community analyses assess microbial community structure using metrics like alpha and beta diversity, unveiling richness, evenness, and composition insights.

Libraries are prepared using the QIAseq 16S/ITS Panels (Qiagen), developed for sequencing 16S rRNA and ITS regions on Illumina platforms and to perform a parallel profiling of bacterial and fungal communities.