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iSeq 100 Loading

iSeq System Sequencing Guide

Genomics Platform

Last Updated: 11/13/2023

A step-by-step guide�

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Preparing & Unboxing Your iSeq

Detailed unboxing instructions

UPS or Surge Protector Recommended

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Preparing & Unboxing Your iSeq

Temperature: 15°C to 30°C (22.5°C ± 7.5°C)
Humidity: Non-condensing 20%–80% relative humidity
Altitude: Less than 2000 m (6500 ft)
Air Quality: Pollution degree rating of II Ventilation: Up to 2048 BTU/hr @ 600 W
For Indoor Use Only

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Preparing & Unboxing Your iSeq

Illumina Site Prep Guide

Illumina Sequencing System Guide

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iSeq Anatomy

iSeq 100 Sequencer

Flow cell

Cartridge

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iSeq Intro

Undenatured libraries are loaded directly into the cartridge

iSeq uses a patterned flow cell like the NovaSeq

Validated up to 150 cycles paired-end (322 maximum cycles)
Cannot accommodate custom primers

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iSeq Chemistry

The iSeq has a one dye and two chemistry step system. It takes two images for every cycle to determine base calling (max 322 cycles).
G is the darkest color on the instrument
Avoid “G” in the first two bases in indices/barcodes

Figure caption - One-Channel SBS Chemistry—(A) One-channel SBS chemistry features two chemistry steps and two imaging steps per sequencing cycle using nucleotides that can be labeled or unlabeled depending on the chemistry step. (B) The base call is determined by the signal pattern across both images.

Each sequencing cycle includes two chemistry steps and two imaging steps. The first chemistry step exposes the flow cell to a mixture of nucleotides that have fluorescently labeled adenines and thymines. During the first imaging step, the light emission from each cluster is recorded by the CMOS sensor. The second chemistry step removes the fluorescent label from adenine and adds a fluorescent label to cytosine. In both chemistry steps, guanine is dark (unlabeled). The second image is recorded. (B) The combination of Image 1 and Image 2 are processed by image analysis software to identify which bases are incorporated at each cluster position. This sequencing cycle is repeated "n"times to create a read length of"n" bases

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Patterned Flow Cell

The patterned flow cell is loaded by diffusion so small molecules are favored to find oligo binding sites on the flowcells.
The ExAMP chemistry is an amplification by exclusion process such that the first molecule to enter the well of the patterned flow cell is amplified and excludes other molecules from binding.
Therefore every adapter dimer will fill a well and give an unproductive read.

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Before Loading...

Thaw Reagent Cartridge

Overnight in 4^oC or
6 hours in room temperature water bath

Take out flow cell from 4^oC

At least 30 minutes before loading

Dilute library to loading concentration (110 pM or optimized concentrations)

Spike in 1% PhiX (default)

Invert the cartridge 5 times then tap gently on table
Insert flow cell into slot (B) in cartridge
Carefully pierce foil with a pipette tip. Do not touch bottom of loading well
Load 20µL of library into cartridge (C)

Flow cell

Cartridge

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Loading Guide Sheet for iSeq

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How to Make a Sample Sheet for iSeq

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Sample Sheet Breakdown

Header section is mandatory but filling in each field is optional.

KEEP!

Manifest section is not necessary. Could delete these lines off sample sheet.

KEEP!

Reads section is mandatory

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Sample Sheet Breakdown (cont.)

Setting section is not necessary.

Data section is mandatory. However, not all fields are necessary. Be sure to fill out: Sample_ID, Sample_Name, Description, I7_Index_ID, Index, I5_Index_ID, index2

NOTE: In any part of the sample sheet: spaces, special characters and commas are not allowed.

Illumina’s sample sheet v2 format specifications

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Pre-run checks will take ~15 minutes. Run starts automatically after the checks are completed.

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iSeq Settings

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iSeq settings and options

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About

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System Check:

Remember to put in system test cartridge + flow cell.

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System Settings

Using Express Settings will use Local Run Manager to create runs. (Different from training)

To set up runs like we do in training, please select Set up Manually and in the Local Run Manager setting choose Use Manual Mode. (Next Slide)

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Set output folder to write to remote server or locally. Data is saved in two places, one copy in the output folder (defined here) and another in the local drive.

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Local copy on computer. Our sequencer also writes to our remote server.

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Process Management:

Can delete runs here if your computer is running out of disk space

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iSeq Troubleshooting

Looking for top unknowns and re-demultiplexing

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Basespace Troubleshooting → Looking for undetermined reads

>Select Analysis

OR

>Go to Projects > Select appropriate Project > FASTQ Generation….

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Looking at top unknowns

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Open in text editor:

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Re-demultiplex with updated Sample Sheet

You have to own the run in order to requeue analysis.
To edit samplesheet and requeue analysis(link):

Go to run
Go to samplesheet tab
Status (right hand side) -> Requeue -> Samplesheet
Edit sample sheet

Easier to update samplesheet in excel first, copy csv as text file and copy/paste in new samplesheet
Can change sample name to differentiate between two demultiplexing

Queue analysis

There is a limit on how many times an analysis can be requeued. If re-demultiplexing is required again, please contact Illumina for assistance.

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Examples of iSeq runs on BaseSpace

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Example of a Run with Fluidic Issue (link)

Normal

Abnormal spikes

Normal

Abnormal Basecalling

PhiX Alignment and Error Rate (usually <2%)

In this case, the sequencer had a fluidic issue at cycle 105. The first 105 cycles have normal intensity and % base readouts, then becomes sporadic. Error rate on PhiX control is also a good indication of machine issues as error rate should be well under 2%.

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Example of a Underclustered Run

% Occupied: 40-70%

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Example a Overclustered Run

% Occupied: 97.1-98.2

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Questions?

Illumina Tech Support

Phone: 1-800-809-4566 (USA) /1-858-202-4566 (International)
Email: Techsupport@illumina.com

CZB SF Sequencing Platform