Sequencing

Cluster generation, sequencing, and analysis comprise sequencing on the iSeq 100. Each step occurs automatically during a sequencing run. Depending on system configuration, further analysis is performed off-instrument after the run is complete.

• Cluster generation—Library is automatically denatured into single strands and further diluted onboard the instrument. During cluster generation, single DNA molecules are bound to the surface of the flow cell and amplified to form clusters.
• Sequencing—Clusters are imaged using one-dye chemistry, which uses one fluorescent label and two imaging cycles to encode data for the four nucleotides. The first imaging cycle detects adenine (A) and thymine (T). A chemistry cycle then cleaves the dye from A and simultaneously adds a similar dye to cytosine (C). The second imaging cycle detects C and T. After the second imaging cycle, Real-Time Analysis software performs base calling, filtering, and quality scoring. This process is repeated for each cycle of sequencing. For more information on one-dye chemistry, refer to Base Calling.
• Analysis—As the run progresses, the control software automatically transfers base call files (*.bcl) to the specified output folder for data analysis. The method of data analysis depends on application and system configuration.

Loading volume is 20 µl. Loading concentration varies by library type and cartridge.

Library Type

Loading Concentration (pM)

1 nM Library Volume (µl)

RSB Volume (µl)

100% PhiX (for a PhiX-only run)

100

10

90

AmpliSeq Library PLUS for Illumina

50

5

95

Illumina DNA Prep

100

10

90

Illumina DNA Prep with Enrichment

75

7.5

92.5

Nextera XT DNA

150

15

85

TruSeq DNA Nano

150

15

85

TruSeq DNA PCR-Free

100

10

90

Illumina DNA PCR-Free Prep

120

12

88

For other library types, Illumina recommends 50 pM as a starting loading concentration. Optimize this concentration over subsequent runs to identify a loading concentration that consistently yields data that meets specifications.

Loading concentrations that are too high or too low result in suboptimal clustering and run metrics. For more information, refer to the Cluster Optimization Overview Guide (document # 1000000071511).

For each read, enter at least 26 cycles and at most 151 cycles to optimize data quality. The exact number of cycles depends on your experiment.

The minimum and maximum cycle numbers include an extra cycle. Always add one cycle to the desired read length to correct the effects of phasing and prephasing. Read length is the number of sequencing cycles in Read 1 and Read 2, which excludes extra cycles and index cycles.

Example run setups:

• For a read length of 36 (single-read), enter 37 in the Read 1 field.
• For a read length of 150 per read (paired-end), enter 151 in the Read 1 field and 151 in the Read 2 field.
• When handling reagents and other chemicals, wear safety glasses, a lab coat, and powder-free gloves. Switch gloves when prompted to prevent cross-contamination.
• Make sure that you have the required consumables and equipment before starting a protocol. Refer to Consumables and Equipment.
• Follow the protocols in the order shown, using the specified volumes, temperatures, and durations.
• Unless a stopping point is specified, proceed immediately to the next step.
• If you plan to thaw the cartridge in a water bath, store the cartridge at -25°C to -15°C for at least 1 day before thawing. A water bath is the fastest of three thaw methods.
1. Put on a new pair of powder-free gloves.
2. Remove the cartridge from -25°C to -15°C storage.
3. If the cartridge is boxed, remove it from the box but do not open the white foil bag.

4. Thaw the bagged cartridge using one of the following methods. Use immediately after thawing, without refreezing or otherwise storing.

Method

Thaw Time

Instruction

20°C to 25°C water bath

6 hours, not exceeding 18 hours
• Use 6 L (1.5 gal) water per cartridge.
• Set a temperature-controlled water bath to 25°C or mix hot and cold water to achieve 20°C to 25°C.
• Face the bag label up, submerge the cartridge completely, and apply ~2 kg (4.5 lb) weight to prevent floating.
• Do not stack cartridges in the water bath unless it is temperature-controlled.

2°C to 8°C refrigerator

36 hours, not exceeding 1 week

Position the cartridge so that the label faces up and air can circulate on all sides, including the bottom.

Room temperature air

9 hours, not exceeding 18 hours

Position the cartridge so that the label faces up and air can circulate on all sides, including the bottom.

Thawing the cartridge in a water bath directly from shipment, when it has been stored on dry ice, can adversely affect performance. Store at -25°C to -15°C for at least 1 day before thawing.

5. If wet from a water bath, dry with paper towels.