Next-Generation Sequencing

The GSF performs all protocols necessary for preparing a biological sample to be sequenced on the following:

Illumina NovaSeq 6000 System

NovaSeq 6000 is supported by NIH Shared Instrument grant 1S10OD030311-01

Illumina NextSeq 2000

For next-generation sequencing studies, the GSF provides the library preparation and sequencing for the following applications:

RNA Sequencing

• Stranded Total RNA Seq
• Stranded mRNA Seq
• smRNA Se

DNA Sequencing

• Whole Genome Seq (WGS)
• Whole Exome Seq (WES)
• cfDNA and FFPE DNA Seq
• DNA Methylation Seq
• PCR Amplicon Seq
• Targeted Gene Re-seq
• CRISPR Screening

Epigenomic Sequencing

• ChIP Seq
• Cut&Tag and Cut & Run
• ATAC Seq
• RNA Immunoprecipitation (RIP) Seq
• Methyl RNA Immunoprecipitation (mRIP) Seq

Single Cell Analysis (individually picked)

• SMART-seq 2
• • SeqWell Rapid Single Cell RNA seq

Single Cell Analysis (10X)

• Single cell RNA seq (3’)
• Single nuclei RNA seq (3’)
• Single cell ATAC seq
• Single cell Multiome seq
• Single cell Immune Profiling (5’)
• RNA FLEX

Spatial Transcriptome Analysis (10X)

• CytAssist Visium
• CytAssist Visium HD

Metagenomics

• 16S V1_V2 sequencing
• 16S V3_V4 sequencing
• Shotgun Seq

Ultra-Deep Sequencing

• TwinStrand duplex seq

DNA/RNA Analysis

• DNA/RNA Quantification
• DNA Fragment Size Check
• RNA Quality Check
• DNA Shearing

Experimental Design

Next-generation sequencing (NGS) experiments and projects are still relatively new technologies and applications for many Principal Investigators. It poses great challenges and investments to design the experiment and understand the logic of downstream bioinformatics analysis. Project planning and experimental design are required. For the sequencing project the GSF works on, we request that prior discussion takes place between the GSF and the project Investigators submitting the samples. Based on the biological questions you are asking, we will work with you to decide on the experiment outline including the number of samples needed (biological replicates and groups), the sample preparation details (DNA-Seq, RNA-Seq, polyA selected or rRNA depleted, enriched procedures or not), the number of reads needed (it affects the pooling scheme), single read or paired-end reads needed, and sequencing length (sequencing module choice, 50PE, 75SR, 75PE,100PE, 150PE), and so on. Please contact us if you are interested in working with us.

Quality Control

We QC every sample we receive. We make judgments of samples based on the time point we have QC data. There are different QC steps involved for the different sample types. Generally, Agilent Bioanalyzer or agarose gel (for genomic DNA), together with Qubit, is used for QC purposes. We will notify the user if there is any issue regarding the quality and quantity of samples and if samples need to be replaced. The tracking of specific QC metrics is recorded in our Wiki LIMS system.

We have developed extensive tools to monitor sequence quality and accuracy; every sequencing run that is performed by the GSF is subjected to quality control evaluation in the form of a report that includes a review of reading output and overall quality metrics, including the Q30 score, percentage of undetermined reads, FastQC result, duplicate rate, mappable rate et al. These QC mechanisms allow GSF to maintain the highest level of sequence quality, which simplifies subsequent analyses.

Quick Facts

Factors you want to know about the new sequencer Illumina NovaSeq 6000

Illumina NovaSeq 6000 is an integrated system that enables higher throughput sequencing at significantly reduced per-base costs and the fastest production scale. NovaSeq 6000 leverages Illumina’s SBS (sequence by synthesis) technology and combines the best features of Illumina sequencers – two-color chemistry and patterned flow cell technology – into a single streamlined operation instrument to enable increasing sequence throughput and higher data generation speed. The instrument-controlled computer performs real-time analysis processing, automatically producing image intensities and quality-scored base calls directly on a four-processor PC in the requested equipment. Optics of NovaSeq 6000 offer high-resolution, high-speed scanning, contributing to high-throughput generation. Each flow cell contains billions of nanowells at fixed locations for even cluster spacing and uniform feature size. NovaSeq flow cells reduce the spacing between nanowells, significantly increasing cluster density. The higher cluster density plus the proprietary exclusion amplification clustering maximizes the number of nanowells occupied by DNA clusters originating from a single DNA template, substantially increasing data output.

There are three significant improvements enabled by NovaSeq 6000 in comparison with most HiSeq platforms:

• Scalability: The NovaSeq 6000 carries four different flow cell types: S Prime (SP), S1, S2, and S4. It generates from 80 Gb and 800 M reads to 3 Tb and 10 B reads of data in single flow cell mode. The output can be up to 6 Tb and 20 B reads in dual flow cell mode in less than two days. SP, S1, and S2 flow cells provide quick and powerful sequencing for most high-throughput applications. In contrast, the S4 flow cell enables high-throughput, cost-effective sequencing that is affordable for applications including whole genome, whole exome, and Hi-C sequencing. With the increasing pace of sequencing output and running speed, NovaSeq 6000 maintains the expected low error rate as other Illumina platforms. Standard workflow allows pooled libraries to load into the entire flow cell. In contrast, the Xp workflow enables labs to load libraries directly into each lane of a NovaSeq 6000 System flow cell: SP, S1, S2 with two lanes separation, and S4 with four lanes separation. 
• Flexibility: The NovaSeq 6000 offers two library loading workflows to separate different projects or methods between lanes. The system is compatible with various Illumina library preparation kits, supporting various applications based on sequencing options.
• Simplicity: The NovaSeq 6000 provides load-and-go reagent cartridges, automated flow cell loading, and onboard cluster generation to minimize hands-on time in the lab significantly.
• Illumina NovaSeq 6000 provides high-quality sequencing data. The Q30 (1 error chance in 1,000) is ≥ 90% for reads of 2X 50 bps, ≥ 85% for reads of 2X 100 bps and 2X 150 bps, and ≥ 75% for reads of 2X 250 bps.

NovaSeq 6000 Sequencing System Specifications

The factors you want to know about the NextSeq 2000 (a)

• Using Illumina’s new XLEAP-SBS technology since just the summer of 2024, NextSeq 2000 offers a fast and easy workflow for any project size and sequencing throughput for numerous popular sequencing applications such as RNA-seq, targeted panels, small RNA-seq, ChIP-seq, ATAC-seq, and single-cell sequencing.
• Based on sample volume and coverage needs, users can choose between four flow cell configurations with P1 100M, P2 400M, P3 1200M, and P4 1800M reads, easily shifting from the low-to higher-throughput processing with each sequencing run.
• Illumina NextSeq 2000 provides high-quality sequencing data, similar to what NovaSeq offers. The sequencing read length from NextSeq ranges from 50bp single read sequencing to 300bp paired-end sequencing.

Sequencing Output Per Flow Cell(a)

NextSeq 1000 and NextSeq 2000 with XLEAP-SBS Chemistry

Reads Passing Filter Per Flow Cell

NextSeq 1000  and NextSeq 2000 with XLEAP-SBS Chemistry

• Illumina NextSeq 2000 provides high-quality of sequencing data, similar to what NovaSeq offers. The sequencing read length from NextSeq ranges from 50bp single read sequencing to 300bp paired-end sequencing.

Sequencing libraries for NovaSeq 6000 and NextSeq 2000

• Sequencing library for NovaSeq 6000 can be sensitive due to patterned flow cell: 1. Primer dimer % < 0.5%; library insert size needs to be < 550bp or total library length < 670bp. Minor “tails” of longer fragments are still suitable.
• A pooling strategy can significantly improve the sequencing efforts. Samples individually barcoded during the library preparation can be multiplexed on a lane. The number of samples that can be pooled per lane depends on the number of reads per sample needed for the following bioinformatics analysis.
• Illumina NovaSeq and NextSeq use two-color chemistry: C base is seen as red, T is green, overlapping in both C and T is A, and G is seen as a dark channel with unlabeled. Thus, try to avoid the first few cycles with only the G base since the G base is identified as a dark channel. At each cycle and color channel, at least one of two nucleotides must be read to ensure proper image registration. It is important to maintain color balance for each base of the index read being sequenced; otherwise, index read sequencing could fail due to registration failure. Follow proper pooling guidelines, depending on the Sample Prep kit you are using.

If you are interested in working with the GSF for the NGS project, please read the Submission of Samples.