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

公式ガイド確認済み

ORDERED_STEPSKIT_VERSION_LOCKEDINDEX_UNIQUENESS_REQUIREDCONTROL_GATEDMANUFACTURER_IFU_OVERRIDES

使用前に自分のassay情報を入力してください

実行順序と合理的な開始条件を整理した下書きです。サンプルのprimer・probe配列は含めていません。必ず自分の標的配列を入力し、実際の酵素・kit・装置の説明書とassay検証データに合わせて条件を確定してください。

LOCK RUN DESIGN

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

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

QUALIFY DNA INPUT

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

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

PREPARE REAGENTS

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

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

TAGMENT DNA

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

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

STOP AND WASH

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

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

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.

温度: Use the kit revision and input-dependent PCR program.試薬: Enhanced PCR mix plus compatible i5 and i7 index adapters条件: USER REQUIRED: verify that no index pair is duplicated in the planned pool.

CLEAN LIBRARIES

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

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

QC AND QUANTIFY

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

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

NORMALIZE AND POOL

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

試薬: Passing indexed libraries and compatible resuspension buffer条件: USER REQUIRED: calculate each library contribution and document exclusions or rebalancing.

SEQUENCE AND REVIEW

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

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

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