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NGS Library Preparation & Sequencing

Complete a PCR protocol for your own assay

The ordered workflow and rational starting conditions are ready. Add your target-specific primers or probe, align the settings with your actual kit, then save the working protocol as Word or Excel.

Editable starter protocols

5 protocols

What do these protocols do?

They select a sample and sequencing workflow, then organize input QC, library preparation, amplification or cleanup, library QC, and sequencing handoff.

Supported NGS workflows

  • • Short-read DNA
  • • Stranded mRNA
  • • 16S amplicon
  • • Nanopore ligation
  • • Single-cell 3'

Scope boundary

Kit-, chemistry-, and instrument-specific conditions stay within the verified source revision and are not extrapolated as universal defaults.

PRT-NGS-STARTER-DNA-PREP-001Short-read DNA

Illumina DNA Prep: genomic DNA to indexed short-read library

NGS Library Preparation & Sequencing · CURATED-PRT-NGS-STARTER-DNA-PREP-001-V1

Official guidance reviewed
ORDERED_STEPSKIT_VERSION_LOCKEDINDEX_UNIQUENESS_REQUIREDCONTROL_GATEDMANUFACTURER_IFU_OVERRIDES

Add your assay information before use

This draft provides an ordered workflow and rational starting conditions. It intentionally contains no sample primer or probe sequence. Enter your target-specific oligonucleotides and finalize every condition against the actual enzyme, kit, instrument instructions, and assay-validation data.

1

LOCK RUN DESIGN

Record the specimen identifiers, genome size, desired depth, paired-end read length, index set, and compatible sequencer before opening reagents.

Condition: USER REQUIRED: confirm sample count, unique index pairs, coverage target, and current kit/instrument compatibility.
2

QUALIFY DNA INPUT

Quantify DNA with a validated fluorometric assay and assess purity and integrity using methods appropriate for the sample type.

Reagent: Purified genomic DNA in a buffer compatible with bead-linked transpositionCondition: Use the current kit guide for accepted mass, volume, and direct-input exceptions; do not infer acceptance from absorbance alone.
3

PREPARE REAGENTS

Bring the required bead-linked transposome and tagmentation reagents to the state specified by the current product guide, mixing without damaging beads.

Temperature: Use the storage and handling temperatures in the current product documentation.Condition: Keep reagent lot, expiration, thaw count, and plate map in the batch record.
4

TAGMENT DNA

Combine qualified DNA with the bead-linked transposome reaction and run the kit-defined tagmentation program to fragment and tag DNA.

Duration: Kit-defined tagmentation time; the current overview describes a 15-minute tagmentation reaction.Temperature: Use the exact current thermal-cycler program for the kit revision.Reagent: Illumina DNA Prep bead-linked transposome and tagmentation components
5

STOP AND WASH

Stop tagmentation and wash the bead-bound library in the prescribed order while keeping beads suspended and preventing cross-well transfer.

Reagent: Current kit stop and wash buffersCondition: Do not substitute buffers or omit washes; follow the plate-safe stopping points in the current guide.
6

INDEX AND AMPLIFY

Add a unique dual-index pair to every sample and perform the limited-cycle PCR specified for the measured DNA input and workflow.

Temperature: Use the kit revision and input-dependent PCR program.Reagent: Enhanced PCR mix plus compatible i5 and i7 index adaptersCondition: USER REQUIRED: verify that no index pair is duplicated in the planned pool.
7

CLEAN LIBRARIES

Perform the prescribed bead cleanup, ethanol washes, drying, and elution without disturbing the pellet or overdrying the beads.

Duration: Use the magnet, wash, dry, and elution times in the current guide.Reagent: Illumina Purification Beads, freshly prepared ethanol, and kit-specified resuspension buffer
8

QC AND QUANTIFY

Measure final library concentration and fragment-size distribution, checking for failed libraries and unexpected adapter-dimer signal before pooling.

Condition: Record assay method, concentration, average fragment size, and the laboratory acceptance decision for every library.
9

NORMALIZE AND POOL

Normalize passing libraries to the chosen molarity and pool only verified index combinations according to the run design.

Reagent: Passing indexed libraries and compatible resuspension bufferCondition: USER REQUIRED: calculate each library contribution and document exclusions or rebalancing.
10

SEQUENCE AND REVIEW

Denature, dilute, and load the pool using the compatible instrument guide, then review run metrics and FASTQ quality before downstream interpretation.

Condition: Instrument-specific loading concentration and quality thresholds must come from the current system and reagent documentation.