Introduction to the NextSeq System

Size: px

Start display at page:

Download "Introduction to the NextSeq System"

Diana Kennedy
5 years ago
Views:

1 Introduction to the NextSeq System 2015 Illumina, Inc. All rights reserved. Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cbot, CSPro, CytoChip, DesignStudio, Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iscan, iselect, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the US and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.

2 Course Objectives By the end of this course, you will be able to: Identify features of the NextSeq System Describe NextSeq Consumables Describe NextSeq Sequencing Technology 2

3 NextSeq 500 Exome, transcriptome, whole genome sequencing in a single run Sample size flexibility Flow cell options (e.g. Mid or High Output flow cells) can be used to select data output levels Scale from a single sample to dozens of samples per run Load & Go workflows Integrated sample-to-results solution: streamlined informatics on-premise or in cloud 3

One System, Two Output Modes High 120 GIGABASES PE150

4 One System, Two Output Modes High 120 GIGABASES PE M CLUSTERS Mid 40 GIGABASES PE M CLUSTERS. 4

5 NextSeq Series Performance Parameters Total times include cluster generation, sequencing and base calling on a NextSeq 500 System. Install specifications are based on Illumina PhiX control library at supported cluster densities (between 170 and 230 k/mm2 clusters passing filter. Actual performance parameters may vary based on sample type, sample quality, and clusters passing filter. The percentage of bases > Q30 is averaged over the entire run. 5

6 NextSeq Sequencing Technology 8

7 Cluster Generation Single DNA Library Amplified Clonal Cluster Library pool loaded into reagent cartridge flows through all 4 lanes of the flow cell Hybridization and cluster generation are automated on the NextSeq system Approximately 5,000 molecules are included in a cluster 9

8 NextSeq 500 Imaging Cycles Two Channel Sequencing only requires 2 images and hence all the data from the four DNA bases is encoded in these two images. This works much like Four Channel Sequencing and uses the same sequencing by synthesis (SBS) method but allows more efficient acquisition of the data. Green Channel Image Red Channel Image 10

9 Four Channel SBS Chemistry: GA, HiSeq, MiSeq Each of the 4 DNA bases emit an intensity of a unique wavelength Collects 4 images During each cycle, each cluster appears in only 1 of 4 images 11

Two Channel SBS NextSeq Series Two channel SBS uses 2 images Builds template over 5 cycles Clusters appearing in green only are T Clusters appearing in red only

10 Two Channel SBS NextSeq Series Two channel SBS uses 2 images Builds template over 5 cycles Clusters appearing in green only are T Clusters appearing in red only are C Clusters appearing in both images are A Clusters not present in either green nor red are G Cluster intensities are plotted and bases are called accordingly 12

11 SBS Evolution 50% Reduction in Imaging Same library prep, clustering and SBS New 2-channel imaging 13

Sequencing Paired End Libraries with Dual Index Read: GAIIx, HiSeq, MiSeq 1 2 3 4

12 Sequencing Paired End Libraries with Dual Index Read: GAIIx, HiSeq, MiSeq Paired End Turnaround Paired End Dual Index Sequencing Utilizes 4 Sequencing Reads 14

13 Sequencing Paired End Libraries with Dual Index Read: NextSeq Index Read 2 (i5) i7 Index 4 Paired End Turnaround Index read 3 i5 Index i5 Index Seq Primer (SBS3rev/BP13) Note on NextSeq Single Read Dual Index Sequencing: Dual-indexed single reads will need to go through paired end turn around During run set up, do not enter cycles for read 2 15

14 NextSeq Hardware 16

15 System enhancements: Optics Power of high throughput apps Size and affordability of a desktop sequencer. 6x MiSeq imaging capability 1/3 Size of a HiSeq 1/3 Capital cost of a HiSeq 6 parallel miniaturized, solid-state optics modules 18

16 System enhancements: Fluidics No Tubes Easily swapped out Reduced Volumes Lower Maintenance 20

17 NextSeq Consumables CARTRIDGE FORMAT AUTOMATED WASH SELF-CLEANING 21

18 NextSeq 500/550 Consumables 22

19 NextSeq 500/550 Kit Contents NextSeq 500/550 Kit Sizes 75 cycles 93 cycles of sequencing 150 cycles 168 cycles of sequencing 300 cycles 318 cycles of sequencing Reagent Cartridge Store at -15 C to -25 C Buffer Cartridge Store at RT Flow Cell Store at 2 C to 8 C Accessory Box Store at -15 C to -25 C HT1: hybridization buffer 23

20 NextSeq 500/550 Kit Options NextSeq 500/550 kits are available in two output types: NextSeq 500/550 High Output Kit Options: 75 cycles, 150 cycles, 300 cycles NextSeq 500/550 Mid Output Kit Options: 150 cycles, 300 cycles 24 Each kit type contains a compatible flow cell and reagent cartridge The buffer cartridge is universal Always check the label markings when you prepare consumables for a run

21 Load & Go Flow cell Color-Coded Icons At a glance ID of kit compatibility HIGH Ships Dry Handle For tactile feedback Plastic Cartridge Protects against drops 25

Flow Cell Design Lanes Pair A Lane Pair B Dual-surface enabled 4 lane format 1 sample/pool is distributed across all 4 lanes Images in lane pairs Lane Pair A: Lanes 1 and

22 Flow Cell Design Lanes Pair A Lane Pair B Dual-surface enabled 4 lane format 1 sample/pool is distributed across all 4 lanes Images in lane pairs Lane Pair A: Lanes 1 and 3 Lane Pair B: Lanes 2 and 4 Reagent Flow: Lane Pair B Reagent flow Lane Pair A: top to bottom Lane Pair B: bottom to top Reagent Flow: Lane Pair A Flow Cell Cartridge 26

23 Flow Cell Format Tile# Rear Camera Section Swath1Swath2 Swath3 Lane 1 L 4 L 2 L 3 Top / Bottom Section Front Nomenclature: 2_ Lane Swath Tile Example: s_1_21304_red.tif = Lane 1, Bottom Surface, Swath 1, Camera 3, Tile 4 27

24 NextSeq 500/550 Reagent Cartridge Preloaded single-use reagent cartridge Contains all reagents required for cluster generation, sequencing and paired-end chemistry Single library or pool of indexed libraries can be loaded into cartridge Contains one wash reservoir for every reagent reservoir Wash reservoir 28

NextSeq 500/550 Buffer Cartridge The NextSeq buffer cartridge is a single-use consumable containing three reservoirs filled with buffers and wash solution Contents of

25 NextSeq 500/550 Buffer Cartridge The NextSeq buffer cartridge is a single-use consumable containing three reservoirs filled with buffers and wash solution Contents of the buffer cartridge are sufficient for one sequencing run Buffer Position Reagent Description 1 BB4: Sequencing buffer 2 BB3: Wash solution 3 BB2: Incorporation buffer 29

26 Spent Reagents Tray When removing or loading the spent reagents tray, be sure to place one hand underneath the tray for support Spent Reagents Tray 30

27 NextSeq Software 31

28 NextSeq Control Software (NCS) NCS guides user through all steps to load consumables and start run Automatic wash is performed after every sequencing run 32

29 NextSeq Data Analysis Primary Analysis Performed by NCS + RTA2 On-instrument computer performs all primary analysis Run Data Output Run folder is sent to a network drive and/or BaseSpace Large BCL files: one per lane and cycle Data is compressed: zipped bcl files and binned Qscores Demultiplexing and bcl to fastq conversion Performed in BaseSpace or in Linux using bcl2fastq 2.0+ No fastq conversion occurs on the instrument 33

30 BaseSpace Integration 34

NCS interface for selection During sequencing run: data can be streamed in real

31 NextSeq BaseSpace Integration Before run: library information and run parameters can be entered in BaseSpace During run set-up: runs set up in BaseSpace appear on NCS interface for selection During sequencing run: data can be streamed in real time to BaseSpace BaseSpace is available on the cloud or through BaseSpace Onsite 35

32 NextSeq Workflows 36

18 Hours Enrichment Panel 12 Mb Region >20x Coverage 2 x 150 bp 20 Samples 26 Hours N/A Whole-Exome >90% at > 10x

33 NextSeq Workflow Options Mid-Output Flow Cell Configuration High-Output Flow Cell Configuration Gene Expression Profiling > 10 M Reads 2 x 75 bp N/A 40 Samples 12 Hours mrna-seq > 40 M Reads 2 x 75 bp 3 Samples 15 Hours 10 Samples 18 Hours Enrichment Panel 12 Mb Region >20x Coverage 2 x 150 bp 20 Samples 26 Hours N/A Whole-Exome >90% at > 10x coverage 2 x 150 bp 3 Samples 15 Hours 9 Samples 29 Hours Human Whole Genome 3 GB Genome > 30x Coverage 2 x 150 bp 1 Sample 29 Hours 37

34 NextSeq Sample-to-Answer Workflow Prep 15 minutes hands-on Sequence 20 minutes hands-on Analyze fully automated, BaseSpace Share secure, unlimited storage 1.5 HOURS HOURS up to 24 HOURS/SAMPLE INSTANTLY 38

35 Questions? 40