最先端のゲノムサービスおよびソリューション

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サンプル調製および輸送

This document provides guidelines on how to prepare, quantify, and submit samples to Novogene. Whether you are submitting DNA or RNA samples, it is essential that the appropriate instructions be followed to enable the successful completion of your project.

Sample Requirements
Pre-Quality Control (QC) Instructions
Demonstrations of Qualified DNA/RNA Samples
Sample Labeling Recommendations
Sample Packing Recommendations
Completing the Sample Submission Form
Shipping Samples to Novogene

SAMPLE REQUIREMENTS

Sample quality directly impacts sequencing quality and subsequent bioinformatics analysis. Therefore, Novogene has extensive sample quality control procedures to ensure submitted samples conform to requirements for downstream processing.

To guarantee the normal processing of your project, samples should meet the standards given below. If your samples do not meet these standards, and you are unable to produce higher-quality samples, please consult with your Novogene Project Manager before shipping your samples.

Notes:

  • Input quantity should be determined by Qubit® instead of by NanoDropTM, and the final quantity and concentration should conform to Novogene’s specifications.
  • Samples not meeting these specifications should be designated as “at risk” by the customer, and will be subject to billing regardless of data quality. Please consult the Project Manager for further details.

1. Human Whole Genome/Exome Sequencing/Target Region Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration Purity
(NanoDropTM/agarose gel)
Strongly recommended Required
Human Whole Genome/ Exome Sequencing/ Target Region Capture Genomic DNA ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 20 ng/μL No degradation, no contamination
Genomic DNA (PCR-free) ≥ 3 μg ≥ 1.5 μg ≥ 20 μL ≥ 20 ng/μL No degradation, no contamination
DNA products of single-cell whole genome amplification ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 20 ng/μL Fragments should be longer than 500 bp
FFPE* ≥ 3 μg ≥ 1.5 μg Fragments should be longer than 1500 bp

* formalin-fixed, paraffin-embedded

2. Plant & Animal Genome Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration Purity
(NanoDropTM/agarose gel)
Strongly recommended Required
≤ 500 bp Insert Genomic DNA ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 50 ng/μL No degradation, no contamination
Genomic DNA (PCR-free) ≥ 3 μg ≥ 1.5 μg ≥ 20 μL ≥ 50 ng/μL
Mitochondrion/
Choloroplast DNA
≥ 1.6 μg ≥ 800 μg ≥ 20 μL ≥ 50 ng/μL
2 Kb Insert Genomic DNA ≥ 30 μg ≥ 15 μg ≥ 20 μL ≥ 50 ng/μL
5 Kb Insert Genomic DNA ≥ 30 μg ≥ 15 μg ≥ 20 μL ≥ 50 ng/μL
10 Kb Insert Genomic DNA ≥ 50 μg ≥ 25 μg ≥ 20 μL ≥ 50 ng/μL
> 10 Kb Insert Genomic DNA ≥ 80 μg ≥ 40 μg ≥ 20 μL ≥ 50 ng/μL

3. Microbial Genome Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration Purity

(NanoDropTM/agarose gel)

Strongly recommended Required
≤ 500 bp Insert Re
Sequencing
Genomic DNA ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 50 ng/μL No degradation, no contamination
Genomic DNA (PCR-free) ≥ 3 μg ≥ 1.5 μg ≥ 20 μL ≥ 50 ng/μL No degradation, no contamination
Metagenomics Library Genomic DNA ≥ 2 μg ≥ 1 μg ≥ 30 μL ≥ 20 ng/μL No degradation, no contamination
Amplicon Genomic DNA ≥ 240 ng (ITS)
≥ 120 ng (16S)
≥ 20 μL ≥ 12 ng/μL (ITS)
≥ 6 ng/μL (16S)
No degradation, no contamination
PCR Products* ≥ 400 ng ≥ 10 μL ≥ 20 ng/μL Fragment size <450 bp, no degradation, no contamination
PCR Products** ≥ 200 ng ≥ 10 μL ≥ 20 ng/μL Fragment size <450 bp, no degradation, no contamination

*One PCR product for one library
**Multiple PCR products for one library (at least 2 different PCR products)

4. Epigenetics Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration Purity
(NanoDropTM/ agarose gel)
Strongly recommended Required
Whole Genome Bisulfite Sequencing Genomic DNA ≥ 6 μg ≥ 3 μg ≥ 20 μL ≥ 50 ng/μL No degradation, no contamination
ChIP-Seq ChIP-Seq DNA ≥ 100 ng ≥ 50 ng ≥ 10 μL ≥ 20 ng/μL Main peak of 100 bp – 500 bp

5. Transcriptome Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration RNA Integrity Number
(Agilent 2100)
Purity
(NanoDropTM
Strongly recommended Required
Eukaryotic RNA-Seq Total RNA (Animal) ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 50 ng/μL ≥ 6.8, smooth base line No degradation, no contamination
Total RNA (Plant and Fungus) ≥ 2 μg ≥ 1 μg ≥ 20 μL ≥ 50 ng/μL ≥ 6.3, smooth base line
Prokaryotic RNA-Seq Total RNA ≥ 6 μg ≥ 3 μg ≥ 20 μL ≥ 50 ng/μL ≥ 6.0, smooth base line
Amplification products from single cells or low input RNA cDNA ≥ 20 ng ≥ 10 ng Peak range: 400 bp – 9000 bp

Main peak: 1200 bp – 2500 bp

6. Small RNA Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration RNA Integrity Number
(Agilent 2100)
Purity
(NanoDropTM
Strongly recommended Required
Eukaryotic small RNA Sequencing Total RNA (Animal) ≥ 6 μg ≥ 3 μg ≥ 20 μL ≥ 50 ng/μL ≥ 8, smooth base line No degradation, no contamination
Total RNA (Plant and Fungus ) ≥ 6 μg ≥ 3 μg ≥ 20 μL ≥ 50 ng/μL ≥ 7.5, smooth base line

7. Long non-coding Sequencing

Library Type Sample Type Amount (Qubit®) Volume Concentration RNA Integrity Number
(Agilent 2100)
Purity
(NanoDropTM
Strongly recommender Required
Eukaryotic Long non-coding RNA Sequencing Total RNA (Animal) ≥ 4 μg ≥ 2 μg ≥ 20 μL ≥ 50 ng/μL ≥ 6.8, smooth base line No degradation, no contamination
Total RNA (Plant and Fungus) ≥ 4 μg ≥ 2 μg ≥ 20 μL ≥ 50 ng/μL ≥ 6.3, smooth base line

8. Pre-prepared library

(1) Library volume requirement:

Data amount (X) Volume requirement*
X < 30 G ≥ 10 μL
30 G ≤ X < 100 G ≥ 20 μL
100 G ≤ X < 400 G ≥ 30 μL
One flow cell ≥ 50 μL
Over one flow cell, please contact your technical support.

*High concentration samples should be diluted before delivery

(2) Library concentration: library concentration quantified by Qubit® 2.0 (Life Technologies): ≥ 0.5 ng/uL

(3) Molarity concentration: 5 – 30 nM;

(4) Insert size: insert + adaptors (120 bp) ± 50 bp (Does not apply to small RNA library). Dilute to 1 ng/µL before checking the insert size by Agilent 2100 Bioanalyzer.

(5) Qualified library standards: qualified insert size, single peak, no miscellaneous peak, no adapters and no primer dimers, Q-PCR verified concentration between 5-30nM.

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PRE-QUALITY CONTROL (QC) INSTRUCTIONS

Customers must provide the results of analysis of sample quality obtained using one of the following methods: Qubit®, NanoDropTM, agarose gel electrophoresis, or Agilent 2100. It is recommended samples be analyzed by Qubit/PicoGreen/gel electrophoresis (with quantity indicator), so that the results will correspond more closely to Novogene QC results. NanoDropTM quantification is NOT recommended. If NanoDropTM is utilized for pre-QC quantification, Novogene strongly recommends that you send more DNA/RNA for processing than the amounts given above.

For gel electrophoresis, the following conditions are recommended:

DNA: 1.0% agarose gel; 1.0% TAE solution; 100V for 40 min
RNA: 1.0% agarose gel; 0.5× TBE solution; 180V for 16 min

Note:
Different electrophoresis conditions may generate a different, and potentially misleading, QC report on your samples. Therefore, it is highly recommended that you adhere to the conditions recommended above for the initial check, and that you provide Novogene with a picture of the gel.

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DEMONSTRATIONS OF QUALIFIED DNA/RNA SAMPLES

1. Demonstration of Markers Used

Novogene utilizes the following molecular size markers for sample quality control testing (Fig. 1).

Demonstrations of Markers
Fig. 1. (A) Trans2K™Plus DNA Marker, (B) λ/HindIII DNA Marker, bp.

2. Demonstrations of DNA sample quality

a) Main types of sample quality
A qualified DNA sample is compared with common types of unqualified samples (Fig. 2):
Novogene Sample Quality
Fig. 2. Examples of DNA quality. (A) Trans2K™ Plus DNA Marker, (B) qualified sample, (C) degraded sample, (D) sample contaminated with RNA, (E) sample contaminated with protein. Red boxes denote areas of contamination.

2.2 Samples with degradation

The gel picture illustrates samples with degradation. Severe degradation can impact the quality of the prepared library and subsequent bioinformatics analysis (Fig. 3):
Degraded Sample
Fig. 3. DNA samples with degradation. Panels A, B, and C demonstrate increasing levels of DNA degradation. M-1, Trans2K™ Plus DNA Marker.

2.3 Samples with RNA contamination

RNA contamination of DNA samples (Fig. 4) can impede the library construction process. It is strongly recommended to digest your DNA samples with RNase before shipping.RNA contaminated samples
Fig. 4. DNA samples contaminated with RNA. Panels A – D demonstrate increasing levels of RNA degradation. Red boxes denote areas of contamination. M-1, Trans2K™ Plus DNA Marker.

2.4 Samples with protein contamination

DNA samples can be contaminated by protein, as illustrated in Fig. 5. It is recommended that you purify protein-contaminated DNA by affinity column. Please note that column purification will lead to some loss of DNA.
Protein contaminated samples
Fig. 5. DNA samples contaminated with protein. Panels A – C demonstrate increasing levels of protein contamination. Red boxes denote areas of contamination. M-1, Trans2K™ Plus DNA Marker.

3. Demonstrations of RNA sample quality

3.1 Main types of sample quality

A qualified RNA sample is compared with common types of unqualified samples (Fig. 6):
Main types of samples
Fig. 6. Examples of RNA quality. (A) qualified sample, (B) samples with protein contamination, (C) samples with degradation, (D) samples with genomic DNA contamination. Red boxes denote areas of contamination. M, Trans2KTM Plus DNA Marker.

3.2 Samples with protein contamination

Protein Contaminated Sample
Fig. 7. RNA samples with protein contamination. Panels A – D demonstrate increasing levels of protein contamination. Red boxes denote areas of contamination. M, Trans2KTM Plus DNA Marker.

3.3 Agarose gel and Agilent 2100 analysis of RNA samples

Agarose Gel Fig 8
Fig. 8. An example of gel electrophoresis (left), and Agilent 2100 (right), results for an acceptable total RNA sample.
Agarose Gel Fig 9
Fig. 9. An example of gel electrophoresis (left), and Agilent 2100 (right), results for a degraded total RNA sample.
Agarose Gel Fig 11
Fig. 10. An example of gel electrophoresis (left), and Agilent 2100 (right), results for an RNA sample with contamination.
Agarose Gel Fig 11
Fig. 11. An example of gel electrophoresis (left), and Agilent 2100 (right), results for a viscous total RNA sample.

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検体のラベリング

  1. 検体のラベルが溶剤に溶解されないよう、そしてチューブから落ちないよう注意します。防水マーカーペンでチューブの管壁または蓋に検体名を書くのを強くお勧めします。あるいは、検体の情報を紙に書き、管壁に貼り付け、そして透明テープでチューブの周りを包み、ラベルを固定します。
  2. 検体輸送の前に、弊社の検体情報シートに記入し、メールにてご添付ください。検体情報シートとチューブのラベルが一致するかどうかをご確認ください。
  3. サンプル名はアルファベット、数字、アンダースコアのみで、8文字以下にご命名ください。数字で始まるのも避けてください。

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検体の包装

  1. DNAとRNA検体に関しては、1.5 mlまたは2 mlスクリューキャップDNase/RNaseフリー遠心チューブをお勧めします。パラフィルムを使ってチューブを封じてください。無水エタノールまたはイソプロパノールなどの有機溶剤は検体の漏れを引き起こし、検体にコンタミをもたらす恐れがあるため、使用しないでください。もし有機溶剤の検体を輸送する場合、スクリューキャップチューブを使い、パラフィルムを10回以上に巻いてください。
  2. 輸送中の破損を回避するために、試料管を50 mlチューブや検体箱にお入れください。チューブを容器内で固定させるために、緩衝材をお使いください。
  3. RNA検体はドライアイスで、ゲノムDNA検体はアイスバッグで、唾液検体は常温でご送付ください。
  4. 弊社のQC基準に準ずるため、96ウェルプレートおよびPCRストライプチューブはご送付なさらないでください。弊社が受け入れ可能なのは1.5 mlまたは2 mlのチューブです。

Fig12-sample-packing
Fig. 12. 検体輸送時OKとNGのチューブ

検体情報シート

検体情報シートはプロジェクトごとにご提出ください。要求されるすべての情報を丁寧にお書きください。完成した電子ファイルをメールで弊社の営業担当者に提出し、電子ファイルを印刷して、検体と同封してご送付ください。 (Fig. 13)
Sample Form
Fig. 13. 検体情報シートの例

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検体の輸送

免責事項:以下の情報は、「非規制物質」に分類されたサンプルを当社施設に出荷するための推奨事項にすぎません。この文書が作成された時点で、gDNA / total RNAが国際航空運送協会(IATA)の梱包指示書によって診断検体と定義されていないため、特別な梱包要件は記載されていません。 規制の継続的な変化に、規制当局のコンプライアンスを確実にするため、常に安全局及び/または出荷部門に確認する必要があります。

    1. すべての検体は当社の品質基準に満たしており、上記のガイドラインに従って調製され、包装されていることをご確認ください。
    2. 検体輸送の前に、Novogeneの担当者に連絡し、必要な書類をご送付ください。
    3. 信頼性の高い国際輸送会社をお選びください。弊社はFedEx (fedex.com)、UPS (ups.com)、DHL (dhl.com)、TNT (www.tnt.com)、USPS (www.usps.com)、World Courier (www.worldcourier.com)などをお勧めします。事前に輸送会社がDNAまたはRNA検体を香港まで輸送できるかどうかをご確認ください。
    4. 検体輸送オプション:
DNA 凍結乾燥のDNA粉末
アイスバッグ(2 °C-8 °C)
コールドチェーン郵送システム(2°C -8 °C)
DNA Stable (Liquid format, Biomatrica)
ドライアイスバッグ (-60 °C – -80 °C)
RNA 凍結乾燥のRNA粉末
エタ沈後ドライアイスでの輸送
RNA stable (Biomatrica)
ドライアイスバッグ(-60 °C – -80 °C)

注意事項:

a. RNA検体に関してはドライアイスでの輸送を強くお勧めします。ほかの輸送方法は不純物の混入やRNAの微量分解をもたらしかねません。

b. ドライアイスやアイスバッグの必要量は季節(すなわち、常温)、輸送時間および発泡スチロールの箱や容器の厚さによって異なります。輸送時間の見積もりについては、お近くの宅配便局にお問い合わせください。 通常、ドライアイスは一日あたり5キログラムの速度で消耗されます。

  • サンプル郵送先住所:
  • 現地の国際輸送会社にINVOICEを要求し、検体と一緒にご送付ください。INVOICEは以下の内容が含まれます。 INVOICE (commercial invoice, customs invoice, or pro-forma invoice) as required for customs, and include it with the shipment. Please complete the INVOICE as below:

    a. RNA or DNA Samples for Research Use Only
    b. Non-Dangerous, Non-Infectious
    c. No Commercial Value, Value for Customs Only
    d. 税関申告価格を1 USDとお書きください。
    e. 検体の数量と概ねの体積をお書きください。
    f. 容器の種類をお書きください。

    Sample Invoice
    Fig. 14. Courier INVOICE Example

    注意事項: INVOICEに検体の由来や包装方法を書かないでください。"human, tissue, cell, blood, blue ice, dry ice"などの言葉を書かないでください。“RNA or DNA Samples for Research Use Only”のみご記入ください(上に示した図のように)。輸送状に弊社の社名を伏せてください。

    (1)検体情報シートと(2) Qubit/Nanodrop/電気泳動/Agilent 2100などのQC結果(もしあれば)を検体と同封してください。DNAとRNAの検体を以上の方法で包装し、以下の住所までご送付お願いします(香港には郵便番号がございません):

    GARY CHAN
    Novogene Co., LTD
    Lot NO.3719, DD104, Kam Pok West Road, Tai Sun Wai, Yuen Long, N.T, Hong Kong
    +852 34859221, 26121032, 26121828
     

    • 検体情報シートと発注書(PO)を弊社の担当者にお送りください。メール表題に郵送サンプルの見積番号をお書きください。それに検体が滞りなく弊社まで到着されるために、輸送会社と追跡番号をお知らせください。
    • 検体が弊社に到着した後、-80 °Cの冷蔵庫に保存されます。プロジェクトマネジャーはプロジェクトの進捗状況を定期的に報告します。

     

    Qubit is a trademark of Life Technologies and Thermo Fisher Scientific.
    NanoDrop is a trademark of NanoDrop Technologies LLC.
    Agilent 2100 Bioanalyzer is a trademark of Agilent Technologies.
    Trans2K Plus is a trademark of TransGen Biotech.

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