Fragment Analyzer – SS NGS Fragment 1-6000 bp

Method: DNF-473-33 - SS NGS Fragment 1-6000bp.mthds

Instrument: Agilent/Advanced Analytical Fragment Analyzer (33 cm capillary array)

Analysis Mode: NGS

1. Document Control

• SOP ID: FA-NGS-1-6000bp-33cm
• Version: 1.0
• Effective Date: __________
• Prepared by: __________
• Approved by: __________
• Review cycle: 12 months

2. Purpose

To define the procedure for running standard-sensitivity NGS fragment analysis on the Fragment Analyzer using the DNF-473-33 method. This method resolves DNA fragments in the 1–6,000 bp range with standard sensitivity, suitable for:

• QC of NGS libraries (amplicon, PCR-cDNA, shotgun)
• Assessment of size selection efficiency (e.g. AMPure bead selection)
• Verification of library size distribution prior to sequencing
• Quantification of library concentration

3. Scope

Applies to all operators performing NGS library QC or fragment analysis in the 1–6,000 bp range using the DNF-473 kit on a Fragment Analyzer.

4. Responsibilities

• Operator: Execute this SOP, record run metadata, verify QC, and report issues.
• Lab lead: Ensure training, instrument maintenance, and SOP review.

5. Safety

• Wear lab coat, safety glasses, and disposable gloves.
• Handle gels, buffers, and plates per SDS.
• Avoid contact with high voltage areas during operation. Only open instrument when software indicates safe.
• Dispose of consumables as per local regulations.

6. References

• Fragment Analyzer user manual.
• DNF-473 SS NGS Fragment 1-6000bp kit instructions.
• Local laboratory policies for data retention and equipment maintenance.

7. Materials and Equipment

Instrument and configuration

• Fragment Analyzer with 33 cm capillary array.
• Array serial and usage count recorded before each run.

Reagents and consumables

• SS NGS Fragment 1-6000bp gel cartridge compatible with DNF-473-33.
• Buffer reservoirs and rinse solution as per kit.
• 96-well plate(s) and plate seals or caps.
• DNA ladder (supplied with kit or compatible sizing standard).
• Nuclease-free water and low-retention tips.

Software

• ProSize 3.0 or later for data analysis and PDF report generation.

8. Sample Requirements

• DNA libraries in TE, EB, or nuclease-free water.
• Recommended input: 0.2–50 ng/µL (standard sensitivity range).
• Minimum volume per well: as per plate format (typically 2 µL sample + diluent).
• Avoid EDTA >5 mM, high salt, or detergents that interfere with electrokinetic injection.
• For very low concentration libraries (<1 ng/µL, e.g. post-bead-selection), results may show concentration but no resolved peaks — this is expected and the TIM value is still informative.

9. Pre-Run Checks

1. Inspect capillary array for leaks, kinks, or salt crystallisation. Record array serial and usage count.
2. Confirm instrument details in software:
   • Device Serial: record
   • FA Version: record
3. Verify consumables:
   • Correct gel loaded and Gel Selection: Gel 1.
   • Fresh buffer and rinse trays in correct positions.
4. Equilibrate reagents to room temperature if required by kit.
5. Prepare plate map with samples, ladder, controls, and blanks.

10. Plate Preparation

1. Thaw and mix reagents gently. Spin down briefly.
2. Aliquot ladder into designated well (typically position 12 of each row).
3. Aliquot samples into designated wells per plate map.
4. Include at least one blank (nuclease-free water) for baseline reference.
5. Seal the plate. Briefly centrifuge to remove bubbles. Keep sealed until loading.

11. Instrument Setup (Software)

Set parameters as follows:

Note: The DNF-473 method uses shorter separation time (40 min) and higher separation voltage (8 kV) compared to the HS Large Fragment method (DNF-464: 60 min, 5 kV), reflecting the smaller target size range.

12. Run Procedure

1. Load the sealed sample plate into the instrument.
2. Confirm tray positions and liquid levels.
3. In software, confirm the method parameters match Section 11.
4. Start the run. Observe for the first 2–3 minutes for abnormal current, leaks, or errors.
5. Allow the 40 min separation to complete.

13. Data Analysis (ProSize)

1. Open the completed run in ProSize 3.0.
2. Verify ladder sizing is correct (expected peaks at kit-specified positions).
3. For each sample, review:
   • Electropherogram — peak shape, baseline, and resolution
   • Total Concentration (ng/µL) — integrated from all non-marker peaks
   • TIM (nmol/L) — total integrated molarity
   • Main peak size (bp) — dominant fragment size
   • Number of peaks — indicator of library complexity/purity
4. Export PDF report containing per-sample peak tables, electropherograms, and summary metrics.

14. Interpretation Guide

Typical Results for ONT PCR-cDNA Libraries

15. Quality Control and Acceptance

• Ladder wells exhibit expected profile and sizing.
• Baseline is stable with distinct peaks where applicable.
• Migration time consistent across capillaries.
• If any criterion fails, document, recondition, and repeat controls before analysing unknowns.

16. Troubleshooting

17. Maintenance

• Perform Full Conditioning at the start of the day or as required by kit.
• Replace gels and buffers per shelf-life and use counts.
• Track Array Usage Count at each run and follow vendor guidance for replacement.
• Clean trays weekly or after spills. Inspect electrodes and stage area.

18. Records to Retain

For each run record:

• Date, time, and operator
• Device serial and FA software version
• Array serial and usage count
• Method name and key parameters
• Plate map and sample IDs
• File path to raw data and PDF reports
• QC outcomes and any deviations

End of SOP