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  • Simplified genome wide detection of genetic variants with NEXTFLEX Rapid XP V2 library prep kit.
Simplified genome
Application Note

Simplified genome wide detection of genetic variants with NEXTFLEX Rapid XP V2 library prep kit.

Whole Genome Sequencing (WGS) is a powerful tool that can detect a broad spectrum of genetic variations, from SNP to large structural variants, across the entire genome of an individual. With decreasing sequencing costs, WGS is becoming a viable option for population-wide screening for specific disease risk, including expanded carrier testing, pharmacogenomic variants, and polygenic risk scores, for example. Additionally, WGS is also gaining interest as a complementary research tool in the context of newborn screening, a cornerstone of pediatric healthcare.

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Introduction

Library prep can be intimidating for labs without extensive NGS experience. The NEXTFLEX™ Rapid XP V2 NEXTFLEX Rapid XP V2 DNA-Seq Kit NEXTFLEX Rapid XP V2 DNA-Seq Kit  Library prep kit incorporates a streamlined protocol to simplify library prep while delivering robust results. Pooling with the NEXTFLEX UDI barcodes NEXTFLEX UDI Barcodes (10NT, 1-24) NEXTFLEX UDI Barcodes (10NT, 1-24)  enables extreme multiplexing to reduce sequencing costs. Additionally, a complete automated workflow is available for labs seeking to increase throughput and minimize human error. Using cell line-derived reference standards with known mutations, we conducted a proof-of-concept study to evaluate the suitability of the NEXTFLEX Rapid XP V2 library prep kit for detecting germline mutations. These standards offer a controlled means to assess the performance of our WGS workflow, confirming its ability to identify genetic variants in sample material. This study also highlights the potential of the NEXTFLEX Rapid XP V2 kit for comprehensive genomic analysis in research applications.

Methods

Briefly, 25 ng of Mimix™ Male Genomic DNA (HD865) reference standard was used as input for whole genome library preparation. 8 replicates were made. Libraries were made using the NEXTFLEX Rapid XP V2 DNA-seq kit NEXTFLEX Rapid XP V2 DNA-Seq Kit for Metagenomics NEXTFLEX Rapid XP V2 DNA-Seq Kit for Metagenomics  and the NEXTFLEX Unique Dual Index Barcodes NEXTFLEX Unique Dual Index Barcodes (8NT index, 1-96) NEXTFLEX Unique Dual Index Barcodes (8NT index, 1-96)  according to manufacturer’s instructions. The technicians that prepared libraries are experienced in molecular biology but had never used this library prep kit before. Once libraries were prepared, they were quantified using the Thermo Scientific® Qubit® assay, pooled and run on a S2 flow cell on Illumina® NovaSeq® 6000 platform at 2x150 bp read length. Analysis was performed using a custom script, using the VCF file provided for the Mimix reference standards and GRch38 build as reference.

mimix data gc bias

Figure 1: Normalized coverage across varying %GC content for the 8 replicates analysed. The minimal GC bias observed indicates a high degree of coverage uniformity in the data generated.

The Mimix Male Genomic DNA reference standard contains 2,663 variants with AF<10% distributed across multiple regions, including 3’UTR, introns etc. Of those, 269 are missense variants, and they are distributed across 149 genes, all of them relevant in the context of newborn screening. Missense variants are particularly significant in genome-wide screening as they result in amino acid substitutions, directly impacting protein function. For this reason, we focus our attention to them in this test. These group of variants comprises mutations classified as benign, uncertain significance and pathogenic.

Results

On average 626 M reads per sample were generated after sequencing. 98.3% aligned to human genome, corresponding to an average genome coverage of 56x. Coverage uniformity is important to ensure reliable and efficient identification of genetic variation across the genome. To assess the uniformity of coverage distribution in the data, we looked at the GC bias across the 8 replicates (Figure 1).

Concordance between expected genotype from Mimix and observed genotype was 100% for the 269 variants studied. The table below lists some of those variants indicating their ClinVar number, the reference and alternative allele, and the average coverage obtained across the 8 replicates for reach of the alleles.

Table 1: Detail of the 15 first variants analysed, all of them concordant with the expected genotype. A full list of the 269 variants can be provided upon request. 
 

Gene dbSNP ID ClinVar Alt(%) Ref Alt Ref coverage Alt coverage
HPS1 rs58548334 175335 69.20% A G 21.8 47.9
RBM20 rs942077 53184 68.00% G C 25.8 53.5
BAG3 rs3858340 53943 48.80% C T 24.6 46.1
ECHS1 rs1049951 370659 100.00% G A 0.0 26.9
ECHS1 rs10466126 1245298 100.00% A G 0.0 31.3
DCLRE1C rs12768894 253714 61.80% T C 19.1 20.1
CDH23 rs779974496 1481561 45.50% C T 26.8 23.0
CDH23 rs1449510921 1319554 48.80% G A 22.9 25.9
CDH23 rs1227065 55102 100.00% A G 0.0 49.8
CDH23 rs1227051 55120 100.00% G A 0.0 53.3
CDH23 rs41281330 55122 45.80% G A 27.1 21.5
CDH23 rs11592462 55162 40.00% C G 28.4 26.1
BMPR1A rs11528010 50221 72.20% C A 27.1 21.0
DBT rs12021720 134332 100.00% T C 0.0 51.1
ATM rs1801516 133907 31.80% G A 30.3 30.4
The variants reported were inspected using IGV across the 8 replicates
Visualization of rs1801516

Figure 2: Visualization of rs1801516, located on the exon 37 of ATM (chr 11) and leading to the change p.Asp1853Asn

Conclusion

WGS is increasingly recognized as a pivotal tool for comprehensive screening across diverse applications. Our evaluation using Mimix reference standards demonstrates that NEXTFLEX Rapid XP V2 library preparation kit delivers uniform data coverage and enhanced variant detection accuracy, even when implemented by first-time users. These results demonstrate that laboratories can confidently adopt this robust solution regardless of their prior NGS experience, supporting reliable and consistent genomic analysis with minimal technical barriers.

Featured products

Robust library prep kit that generates high-complexity libraries for NGS on Illumina and Element platforms.

NEXTFLEX Rapid XP V2 DNA-Seq Kit

&lt;p&gt;NEXTFLEX™ Rapid XP V2 DNA-Seq Kitは断片化ベースの全ゲノムシーケンシング（WGS）ライブラリー調製ワークフローで、最短2.5時間でDNAからシーケンス用ライブラリーを調製できます。NEXTFLEX独自のノーマライゼーションビーズが、ライブラリー濃度をライブラリー調製プロセス中に均一化するため、別途の定量化や手動プーリングが不要です。&lt;/p&gt; &lt;p&gt;このプロトコルは、100 pgから1 µgのサンプルに対応し、250 ng以上に対応するPCR-freeオプションを提供してライブラリーの複雑性を保ち、GCバイアスを最小限に抑えます。結果的に得られるライブラリーは、Illumina®およびElement Biosciences™ AVITIプラットフォーム上で均一なカバレッジと低い重複率を示し、全ゲノム、メタゲノム、およびその他の研究用途での確実なバリアント検出を可能にします。&lt;/p&gt; &lt;p&gt;自動化対応の試薬、高いインデックス能力（最大1,536 UDI）、合理化されたクリーンアップにより、あらゆる規模で迅速で再現性のあるDNA-seqの結果を提供します。&lt;/p&gt;
  • シングルチューブ酵素断片化、エンドリペア＆Aテーリング - 最短2.5時間でDNAをシーケンシング対応ライブラリーへ
  • 250 ng以上のサンプルに対応するPCR-freeオプションおよび100 pg - 1 µgの広範囲なサンプル濃度に対応したPCR増幅
  • NEXTFLEXのノーマライゼーションビーズ - ライブラリー後の定量化や手動プーリングを省略可能
  • 低濃度のアダプターダイマーの形成、および均一なGCカバレッジで確実なバリアント検出
  • 大規模な多重化研究に対応する最大1,536 UDI利用可能
  • 96 UDI-UMIバーコードセットにより、希少バリアント検出の分子誤差補正が可能
  • Revvity Automation NGSワークステーションで検証済みの自動化対応ワークフロー
  • 各ロットを徹底的にテストすることで品質を保証しています。

NEXTFLEX Rapid XP V2 DNA-Seq Kit は、速度、データ品質、スケーラビリティを融合しています。酵素断片化によりワークフローが合理化され、独自のノーマライゼーションビーズがQCステップ全体を省略し、オプションのPCRフリー経路がライブラリーの複雑性を保ちます。サンプルを少量処理する場合でも、自動分注機上で96ウェルプレートを処理する場合でも、Illumina®およびElement AVITI™シーケンシングに対応した一貫性のある偏りのないライブラリーを作成します。

Learn more
Adapters for NGS sequencing and multiplexing compatible with Illumina and Element systems.

NEXTFLEX UDI Barcodes (10NT, 1-24)

&lt;p&gt;Multiplex up to 1,536 samples on a single flow cell with NEXTFLEX™ Unique Dual Index Barcodes (10NT UDIs). Each kit ships with up to 1,536 pre-validated i5/i7 index pairs that eliminate index hopping and are compatible with Illumina platforms such as the NovaSeq® and Element’s AVITI™ systems. Off-the-shelf plate sets (containing between 16 to 1,536 unique adapters) let you start small and scale to population-level projects without requiring custom ordering or re-balancing.&lt;/p&gt;
  • Up to 1,536 unique i5 / i7 pairs (10NT) ready to ship
  • Compatible with PCR-free and PCR-amplified workflows
  • ≥ 3-base Hamming distance across the entire set for robust error-correction and balanced base diversity on 2- & 4-color chemistries
  • Designed to minimize index hopping that can be present with patterned flow cells.
  • Compatible with Revvity library prep and TruSeq style, ligation-based workflows
  • Every lot is functionally validated and tested for index purity by sequencing in an ISO 9001 facility
  • Optional NEXTFLEX Universal Blockers boost on-target hybrid-capture efficiency when used with these UDIs.
Learn more
Adapters for NGS sequencing and multiplexing compatible with Illumina and Element systems.

NEXTFLEX Rapid XP V2 DNA-Seq Kit for Metagenomics

&lt;p&gt;The NEXTFLEX™ Rapid XP v2 DNA-Seq Kit for Metagenomics offers fast, fragmentation-based library prep for whole genome sequencing of microbial communities for sequencing on Illumina® and Element® platforms. Libraries are ready in ~ 2.5 hours, and proprietary normalization beads eliminate the need for quantitation or manual pooling. The protocol supports 100 pg to 1 µg input, with a PCR-free option at ≥ 250 ng. Purchase includes access to Cosmos-Hub®, providing validated pipelines for WGS metagenomics analysis, including taxonomic and functional outputs.&lt;/p&gt;

The NEXTFLEX Rapid XP v2 DNA-Seq Kit for Metagenomics combines fast, automation-ready library prep with built-in normalization and integrated downstream analysis. Designed for whole genome sequencing of microbial communities, purchase includes access to Cosmos-Hub® for streamlined WGS metagenomics interpretation.

  • Single-tube enzymatic fragmentation, end-repair and A-tailing. Libraries in as little as 2.5 hours
  • PCR-free option for ≥250 ng input; full 100 pg–1 µg input range with amplification
  • Integrated NEXTFLEX normalization beads, skip post-library quantitation and pooling
  • Validated Cosmos-Hub access for taxonomic and functional WGS metagenomics analysis
  • CHAMP and KEPLER pipelines support version-controlled, reproducible reporting
  • Up to 1,536 UDIs available for high-throughput multiplexing
  • Automation-validated on Revvity Sciclone™ and Zephyr™ NGS platforms
  • Compatible with Illumina and Element sequencing platforms
Learn more
Adapters for NGS sequencing and multiplexing compatible with Illumina and Element systems.

NEXTFLEX Unique Dual Index Barcodes (8NT index, 1-96)

&lt;p&gt;NEXTFLEX™ Unique Dual Index Barcode（UDI）は、シングルリードまたはペアードエンドリードシーケンシングに使用できるバーコード付きアダプターです。各インデックスは、3塩基以上の編集距離で分離されており、エラー訂正されたデマルチプレキシングを可能にし、パターン化されたフローセル上でのインデックスホッピングを大幅に制限します。これらのバーコードは、TruSeqスタイルのTオーバーハングワークフローに適合し、すべてのIllumina®機器およびElement BiosciencesのAVITI™システムと互換性があります。最大384個のインデックスが利用可能です。各ロットはインデックス純度を保証するためにシーケンスで検証されており、データの完全性を保護し、サンプルごとのコストを低く抑えます。&lt;/p&gt;
  • Illumina®およびElement®シーケンシングプラットフォーム向けの最大384ライブラリーをマルチプレックス化するためのUDIフルレングスアダプター
  • すべてのPCRフリーおよび増幅されたTruSeqスタイルのTオーバーハングライブラリ調製に対応し、交換可能
  • Unique Dual Indexの設計（ハミング距離≥3）は、パターン化されたフローセル上でのインデックスホッピングやサンプル誤割り当てを軽減します。
  • 各ロットはインデックス純度を保証するためにシーケンスでQC確認済み
  • NEXTFLEX Universal Blockersに対応
Learn more

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Mimix標準サンプル

Revvity の Mimix 標準サンプルはすべて細胞株由来であり、分子診断ワークフロー全体にわたってゲノム領域を広くカバーするよう設計されています。

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