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MiSeq Loading

A step-by-step guide

Angela Detweiler, Norma Neff, Honey Mekonen

Genomics Platform

Last Updated: 02/10/2024

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MiSeq System Guide

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MiSeq parameters:

  • 1 - 24 Million reads
  • 2 x 300 bp
  • Max 600 cycles
  • Run 4-56 hrs
  • Sequencer takes four images per cycle
  • Applications: small whole genomes (microbiome), targeted genes, 16S metagenomics
  • Handles low diversity runs well

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MiSeq Chemistry

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  • Sequencer takes four images per cycle
  • Applications: small whole genomes (microbiome), targeted genes, 16S metagenomics
  • Handles low diversity runs well
  • MiSeq uses a non-patterned flowcell

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Illumina Sequencing Technology - Workflow A

  • The i5 index sequence is the forward sequence sequence, as ordered (MiSeq)

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Illumina Sequencing Technology - Workflow B

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P5

I5 index

P7

Read1 Primer

I7 index

Read2 Primer

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Instrument Components

  1. Flow cell compartment
  2. Enclosed optics compartment
  3. Status bar
  4. Touch screen monitor
  5. External USB ports
  6. Reagent compartment

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  1. Reagent chiller
  2. Sipper handle
  3. PR2 bottle
  4. Waste bottle
  5. Reagent cartridge

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MiSeq Kit

Flow cell in buffer (stored in 4°C); requires 2-3x washes with DI, followed by ethanol wipe, and drying of the flow cell with a Kimwipe

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Reagent cartridge �(stored at -20°C)

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Hybridization buffer

(HT1)

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Incorporation buffer (PR2)

(stored in 4°C)

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Sequencing Kits and Loading Concentrations

  • MiSeq V2 (V2 - 12M, Micro- 4M, Nano-1M)
    • Available in 50, 300, 500 cycles kit
    • Our optimized loading concentration: 10pM in 600µL
    • Cluster Density Goal: 1000-1200/mm2

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  • MiSeq V3 (24M)
    • Available in 150, 600 cycles kit
    • Our optimized loading concentration: 12pM in 600µL
    • Cluster Density Goal: 1200-1400/mm2

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Detailed Kit Costs (US Market as of Feb/2024)

Instrument

Kit (cycles)

Kit Cost ($)

Number of Reads PF (M)

Cost p/ Million reads

Yield p/ Run (Gb)

Cost p/ Gb

Run Time (h)

MiSeq

V2 Nano (300)

340.00

1

333.00

0.5

666.0

17

V2 Micro (300)

514.00

4

125.50

1.2

418.3

19

V2 (300)

1,223.00

12

99.50

5.1

234.1

24

V3 (600)

1,790.00

24

72.83

7.6

230.0

56

NextSeq 2000

P2 (300)

3,629.00

400

9.07

120

30.2

29

P3 (300)

6,150.00

1200

5.13

360

17.1

48

NovaSeq 6000

NovaSeq SP (300)

3,075.00

800

3.84

250

12.3

25

NovaSeq S1 (300)

5,381.00

1300

4.14

500

10.8

25

NovaSeq S2 (300)

9,840.00

3000

3.28

1250

7.9

36

NovaSeq S4 (300)

14,760.00

10000

1.48

3000

4.9

44

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Before Loading

  • Thaw Reagent Cartridge
    • Overnight in 4oC or water bath (room temp ~1 hr)
  • Take out Flow Cell and Incorporation Buffer from 4oC
    • At least 30 minutes before loading to equilibrate to room temperature
  • Invert the cartridge 5 times to mix - avoid making bubbles
  • Carefully pierce foil (well #21) with a pipette tip once ready to load the diluted library into cartridge

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Library Distribution

Library Distributions

  • Average fragment size should be 300-600 bp
  • Fragment that is 300 base pairs long has about 150 base pairs of adapter sequences
    • Long enough to run up to PE 150
  • Adapter Dimers (~140 bp) are bad because they preferentially cluster to the flowcell
    • Results in inaccurate loading
    • A small percent of dimers will take up a significant proportion of reads.

Good Sample

Bad Sample

SPRI Select

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Denature and dilute library

  • Quantify sample pool by bioanalyzer/tapestation and qPCR (if possible)

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  • Loading concentration can be variable between NextSeq 2000 (keep a log)
  • 1-5% PhiX recommended (15-20% for amplicons)

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5µL Sample Pool (4 nM)

2 nM pool

10-12pM Final pool

Denature with 5µL 0.2N NaOH for 5 minutes. Dilute pool to 20pM by adding 990µL of HT1

Further dilute 2 nM pool with RSB and spike in un-denatured PhiX. Final Volume needed is 24uL

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Log into BaseSpace, then click Next.

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Press the white release button to open the flow cell clamp (1). Seat the flow cell and close the clamp (2). Close the flow cell compartment door (3). Once all items , click “Next”.

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Remove/dispose of spent reagents and return the empty waste container. Remove the wash PR2 bottle and load the new buffer. Once all items , click “Next”.

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*Make sure that the sipper handle is down after inserting the PR2 and waste bottle. This will also come handy when troubleshooting.

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The MiSeq requires that the user upload a sample sheet. At CZ Biohub, we download the sample sheet from gDrive to the Sequencer Desktop. The sample sheet name needs to be changed to the reagent cartridge ID (e.g. “MS3046361-600V3”).

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Browse for the location of the file.

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Samplesheet

Make sure to add “Experiment Name, XXXX,,,,,,,,” in line 3 as highlighted in the sample sheet example here. This will name the run on BaseSpace.

Sample sheet example

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Index Reads

  • When preparing a sample sheet for a MiSeq run-
    • I7 index (Index 1) sequence is the reverse complement of the ordered sequence
    • I5 index sequence (Index 2) is the sequence that you ordered

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P5

I5 index

Read1 Primer

P7

I7 index

Read2 Primer

Illumina Sample sheet documentation

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Once the sample sheet is selected, click “Save and Continue”

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Finally remove the the wash cartridge, and slide in the new reagent cartridge. Close the reagent cartridge doors. Once all items , click “Next”.

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Review the run setup information pulled from the uploaded sample sheet.

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If the Read or Index length needs to be updated, go back to the sample sheet, make changes and re-upload the sample sheet.

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Click “Next”.

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The pre-run check will take a few minutes. Once all items , click “Start Run”.

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Cluster density and PF% metrics will become available after 20 and 25 cycles, respectively.

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MiSeq runs can last from 4-56 hrs depending on the kit.

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Perform Wash

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Wash Types

  • Maintenance Wash: Once every month (~90mins)
    • A series of three wash steps with 0.5% Tween20

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  • Stand by Wash: Only if machine will be idle for for more than a week.
    • A series for two washes with 0.5% Tween20

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  • Post-Run Wash: Mandatory after every run with 0.5% Tween20

When troubleshooting, you may need to raise the sippers manually

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Run Options

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Manage Instrument

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Custom Primers

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MiSeq Installation

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