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:

• DNA seq (whole-genome de novo sequencing, whole-exome sequencing, candidate gene re-sequencing, target sequencing, amplicon sequencing)
• RNA seq (Total RNA sequencing, stranded mRNA sequencing, RIP sequencing, CLIP sequencing, SMART-seq2 protocol with ultra-low RNA input and single-cell RNA-seq)
• Small RNA seq
• ChIP seq
• MBDCap DNA seq
• Single-cell sequencing
• 16S based metagenomics
• Targeted gene re-sequencing
• Targeted gene expression
• Cancer gene panel
• CRISPR NGS screening

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 that simplifies subsequent analyses.

Quick Facts

The factors you want to know about the new sequencer Illumina NovaSeq 6000

Illumina NovaSeq 6000 is an integrated system to enable 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 that automatically produces image intensities and quality-scored base calls directly on a four-processor PC, included 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, while the S4 flow cell enables high- throughput cost-effective sequencing affordable for applications including whole genome, whole exome, and Hi-C sequencing. While 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, while Xp workflow enables labs to load libraries directly into each lane of a NovaSeq 6000 System flow cell: SP, S1, S2 with 2 lanes separation, and S4 with 4 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 a wide range of applications, based on sequencing options.
• Simplicity: The NovaSeq 6000 provides load-and-go reagent cartridges, automated flow cell loading, and onboard cluster generation, to significantly minimize the hands-on time in the lab.
• 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 150bps, and ≥ 75% for reads of 2X 250 bps.

The factors you want to know about the NextSeq 2000

• Using Illumina core SBS technology, NextSeq 2000 offers a fast and easy workflow for any project size and sequencing throughput for numerous popular sequencing applications such as exome-seq, RNA-seq, targeted panels, and small RNA-seq.
• Based on sample volume and coverage needs, users can choose between three flow cell configurations with P1 100M, P2 400M and P3 1200M reads, easily shifting from the low-to higher-throughput processing with each sequencing run.

• 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 length of library < 670bp. Minor “tails” of longer fragments are still suitable.
• 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, at each color channel, at least one of two nucleotides needs to 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.