Core Facilities

SCIC Training Web Site

This webpage covers all training/ maintenance  sources for each device. If you have comments on these web page, please email us.

SCIC Device Tutorial

The following accordion part has separate items for each device. 

This item is designed for NextSeq 2000. 

NextSeq 2000 Manual and SOP for standard chemistry, SOP for kits with XLEAP technology

All training documents on the Illumina

Important Links:

Training and Supplementary Documents:

  • Workflow Doc: It provides the speicifications of the device and kits and briefly demonstrates how to perform a run. 
  • Monitoring a run: This file elaborates how to evaluate run results and gives trips on how to optimize a custom library run.
  • Dragen Intro: It provides a background info about the Dragen on NextSeq 2000.
  • Proactive Doc: Proactive monitors all runs in a specific NGS device. The doc summarizes how to use it.

 

Related Videos:

 

 

A demonstration of our new NextSeq 1000/2000 XLEAP-SBS cartridge preparation ‘lift and release’ step

 

 

 

NextSeq2000: Run Quality and Best Practices

 

 

How to remove the components of a cartridge for recycling.

 

 

Introduction to NextSeq 2000

 

 

How to Generate Sample Sheets for Local Run

 

 

How to Generate Sample Sheets for Local Run

 

 

How to requeue analysis for a NextSeq2000 run

 

 

How to power cycle NextSeq 2000

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This item is designed for MiSeq. 

MiSeq Manual and SOP.

Important Links:

The washing steps are used for the maintenance of the device  

Here are the types of washes for MiSeq:

 

Post-Run Wash:  After every sequencing run.

1X wash step: 0.5% Tween 20 wash solution (Wash volume: 17.25 ml)

     a. Add 6 ml wash solution to each reservoir of the wash tray.

     b. Add 350 ml wash solution to the 500 ml wash bottle.

Maintenance Wash: Required every 30 days (instrument will begin warning user that maintenance wash is required at 30 days and it will prevent user from sequencing if maintenance wash has not been completed within previous 45 days).

3X wash steps: 0.5% Tween 20 wash solution (fresh for each wash step) (Wash volume: 51.75 ml).

     a. Add 6 ml wash solution to each reservoir of the wash tray.

     b. Add 350 ml wash solution to the 500 ml wash bottle.


Standby Wash: Prepares instrument for idle mode (if instrument unused for > 7 days), and every 30 days if instrument remains idle.

2X wash steps: 0.5% Tween 20 wash solution (fresh for each wash step) (Wash volume: 46 ml).

     a. Add 6 ml wash solution to each reservoir of the wash tray.

     b. Add 350 ml wash solution to the 500 ml wash bottle.

When the standby wash is complete, the instrument is in standby mode and a message appears on the home screen indicating the status of the instrument.

When the instrument is in standby mode, a maintenance wash must be performed before a sequencing run can be initiated.

Note:

  • If you are running your instruments frequently (2-3 runs/week) It is recommended that the power cycling should happen once a week.
  • If running less frequently than that, please power cycle once in two weeks. If running rarely, please power cycle before each run.

Power Cycle: In every 2 weeks, it is necessary. Please watch the following video.

Training Video:

  • A complete MiSeq run

  • How to denature and dilute libraries and spike in PhiX

  • How to prepare (thawing, mixing, etc.) all reagents (cartridge, buffer, flow cell) before the run

 

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Explore the P2 Solo Training Hub to elevate your sequencing skills. Dive into expert-guided tutorials and unleash the potential of NGS research.

P2 Solo and its SOP.

Details: The P2 Solo is a compact, cost-effective sequencer, offering a more budget-friendly entry point compared to its competitors. Similar to other ONT instruments, it excels in long-read sequencing, spanning from 20 bases to over 4 Mb. Users have the flexibility to run each flow cell independently or simultaneously, allowing for the multiplexing of up to 96 samples in a single run. The P2 Solo supports various applications, including direct RNA sequencing, direct methylation detection, and chromatin conformation capture with Pore-C. When both flow cells are utilized, data output can reach up to 580 Gb, with expected outputs per flow cell as follows: 100-200 Gb for native gDNA reads (read N50 - 25 kb), 50-100 Gb for ultra-long native DNA reads (>50 kb read N50), and >200 Gb for metagenomic samples (read N50 ~ 10 kb).

 

  • How it works

 

  • How to design a NanoPore Sequencing Experiment

Training Video:

  • How to load PromethION Flow Cell

 

  • Extract high quality DNA and RNA

 

  • How to select the right library prep workflow for your experiment

 

  • How to perform tumour and normal sequencing

 

  • How to get started with data analysis

  • Advances in duplex basecalling and how to achieve Q30

More videos related to data analysis: Video 1, link 1, NanoPore 101

Cool topics:

This item is designed for TapeStation. 

Agilent 4200 TapeStation System Manual, SOP, the consumables and reagents

 

  • How to run the TapeStation.

  • How to evaluate the TapeStation results.

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This web page is dedicated to CFX384 Touch Real-Time PCR Detection System located in Single Cell Immunophenotyping Core. 

Although the Maestro & device manual and its SOP contain enough info to operate the device, the following training videos are essential to be able to completely use the device.  

Training Sources for the device and its software called Maestro

  • First set-up

  • How to set up the protocol by using Maestro

  • How to set up the plate by using Maestro

  • How to set up biological and technical replicates in CFX Manager

  • How to analyze data in CFX Manager

Alternative Links:

Quick Guides (pdf documents):

 

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This section is generated for 10X Genomics Controller. 

10X Genomics Chromium Controller Manual and SOP

 Important Links:

Training Videos:

  • Loading Chromium Next GEM Chip G

  • 10X Chromium run

  • Checking the droplets after 10X Chromium run

 

There are a lot of visual experiments in JoVe which is a prier reviewed scientific video journal. For example

 

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NextSeq 2000:

1. What are the manual loading volume and concentrations? 

Normally, the library is automatically diluted and denatured by NextSeq 2000. 

Loading volume is 20 ul.

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For manual dilutions, please use the following numbers: 

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2. What is the cost analysis of MiSeq kits? 

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3. How is the integrated dry cartridge different from the reagents of the MiSeq™ or NextSeq 550 Systems?

The NextSeq 1000 and NextSeq 2000 Systems are referred to as dry instruments because they use cartridges that integrate all fluidic components necessary for amplification and sequencing. In contrast, MiSeq and NextSeq 550 systems contain separate fluidics within the instrument. The integrated cartridge helps to reduce the possibility of cross-contamination. Fluid waste can be purged back into reagent cartridges for easy disposal and multiple plastic components in the cartridge can be easily separated from the waste components and recycled. 

4. Do I need to know the specific flow cell cluster density? (NextSeq 2K)

Patterned flow cells consist of a nanowell substrate with billions of ordered wells. The NextSeq 1000 and NextSeq 2000 Systems use patterned flow cells, which result in a fixed cluster density, even if all nanowells are not occupied. Compared to nonpatterned flow cells, the uniform cluster sizes enable optimal spacing and increased cluster density. In these systems, monitoring the cluster percent passing filter is a better measure of potential cluster occupancy.

5. What are the considerations for switching to the patterned flow cell technology? (NextSeq 2K)

The patterned flow cell technology used by the NextSeq 1000 and NextSeq 2000 Systems is a large improvement over previous technologies due to the ability to avoid overclustering. However, some assay optimization will likely need to take place, especially for custom and third-party library preparation methods. Illumina support specialists are prepared to help every step of the way, from establishing a robust workflow and evaluation process to offering a 1:1 consultation with a field application specialist, or the ability to send samples to an Illumina customer solutions lab.

 

6. What are the storage conditions for NextSeq 2K consumables?

NextSeq 550 System consumables have a 3-month shelf life. NextSeq 1000 and NextSeq 2000 System consumables have a shelf life of 6 months. 

 

7. How to calculate the concentration of PhiX for MiSeq and/or NextSeq runs?

The PhiX Control v3 Library (commonly referred to as PhiX, FC-110-3001) is derived from the small, well-characterized bacteriophage genome, PhiX. It is a concentrated Illumina library (10 nM in 10 µl) that has an average size of 500 bp and consists of balanced base composition at ~45% GC and ~55% AT. Due to its balanced nucleotide composition, the PhiX Control v3 Library is also an ideal sequencing control (typically with ≥ 1% spike-in) for run quality monitoring; e.g. cluster generation, sequencing, and alignment (more info). Please quantify the concentration of PhiX using Qubit before the spike-in. The following converter can be used to convert ng/ul to nM: NanoMolar Converter. 

8. Is it possible to directly download the runs in our BaseSpace to our RCC account? 

Yes, it is. BaseSpace Sequence Hub Command Line Interface (CLI) helps you to quickly download your runs to RCC. Please watch the video and check the guide file. 

9. How to download fastq files from our BaseSpace account to our RCC account? 

Please watch the video and check the guide file. 

10. How to convert mass (ng) per µl DNA to fmol per µl DNA (needed for PromethION Solo Runs)

Please use the following link: https://nebiocalculator.neb.com/#!/dsdnaamt 

 

Under the construction. Soon! 

Supplementaries:

 

Videos:

  • Intro to MinION

  • Intro to MinION M1kC

  • How to run MinION M1kC

  • Priming and Loading your flow-cell

Note: Other videos that explains how to prime and load a flow cell, Video 1, Video 2, 

  • Priming and Loading your flongle flow-cell

 

 

 

 

Data Management:

Data Transfer Methods for Cloud Environments

  1. Globus:

    • A robust file-sharing and transfer service.
    • Users can schedule transfers after data generation by P2Solo or M1kC.
    • Ensures efficient and reliable data movement.

  2. Samba:

    • An easy data management method, but not recommended for privacy reasons.
    • Users can visualize the cloud folder in the directory, transfer data, and remove it afterward.

  3. SFTP (Secure File Transfer Protocol):

    • A secure alternative to FTP.
    • FileZilla, a free and open-source FTP application, facilitates data transfer.
    • Best practice: Transfer data from local folders to cloud-based storage for redundancy, collaboration, and secure analysis access.

Links

Videos:

  • Globus

 

  • Samba

 

  • Filezilla