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NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs)

NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) カタログ番号:E6440

カタログ番号

サイズ

濃度

価格

保存温度

E6440S 96 rxns ¥77,500 -20C
E6440L 384rxns ¥278,000 -20C

製品カテゴリ>グループ

  • NEBNext次世代シーケンサー用ライブラリー調製試薬>Illumina用アダプターオリゴ

特徴

Illumina用 アダプターオリゴ


特長:

・最大 96 プレックス解析(NEB #E6442 と併用すれば最大 192 プレックス)

・ユニークなインデックス・プライマーペアにより、インデックス・ホッピングを低減

・シングルエンド、ペアエンド解析対応

 

Figure 1. アダプターオリゴの付加手順
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NEBのアダプターオリゴは1本鎖DNAがループを形成した構造をとっており、頂点部にウラシル残基を保有します。アダプターライゲーション後にUSER Enzymeを加えて15分間処理することにより、ウラシルが切断されてPCRで増幅可能な断片となります。インデックスやP5、P7配列はPCR反応の際に付加されます。PCRフリーのアプリケーションには対応していません。

 

 

Figure 2: Use of NEBnext Adaptor and Unique Dual Index Primer Pairs substantially increases library yields and minimizes adaptor-dimers
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16 libraries were prepared with 100 ng of Human NA19240 genomic DNA (Coriell Institute), using the NEBNext Ultra II FS DNA library prep kit. Adaptors and primers were from either the IDT® for Illumina® –TruSeq® DNA UD Indexes (Illumina # 20022370), or the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs). After 4 PCR cycles, libraries were quantified on an Agilent® TapeStation® 4000. A) Average library yields for the 8 libraries made using each workflow show 60% higher yield when the NEBNext Adaptor and Unique Dual Index Primer Pairs are used. B) TapeStation traces of 16 libraries show the presence of adaptor-dimer (indicated by the orange arrow) with the libraries made using IDT for Illumina adaptors and primers, which is absent in the libraries prepared using NEBNext adaptors and primers, which also have higher yields.

 

 

Figure 3: Libraries amplified with NEBNext 96 Unique Dual Index Primer Pairs cluster evenly on the Illumina® NovaSeq™ 6000

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96 libraries produced using the NEBNext Ultra II FS DNA library prep kit (Figure 1) and the NEBNext 96 Unique Dual Index Primer Pairs were pooled at equimolar concentrations and sequenced on the Illumina NovaSeq 6000 instrument (2 x 150 bp). The total number of reads from all libraries were summed, and the fraction of the total reads contributed by each library was determined (expected fraction per library=1.04%). All 96 libraries clustered efficiently and were represented at approximately the expected frequency.

 

 

Figure 4: NEBNext 96 Unique Dual Index Primer Pairs amplify libraries with equal efficiency
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Human NA19240 genomic DNA (Coriell Institute) was used to prepare 96 libraries using either the NEBNext Ultra II FS DNA library prep kit, Covaris-sheared DNA with the NEBNext Ultra II DNA library prep kit, the Ultra II DNA library prep kit combined with bisulfite conversion, or the NEBNext Ultra II Directional RNA library prep kit. Libraries were PCR-amplified using the NEBNext 96 Unique Dual Index Primer Pairs to produce libraries containing unique i5 and i7 indices. Library yields were quantified (Agilent® TapeStation® 4200) and normalized by summing the total yield of all 96 libraries and calculating the contribution from each library (expected fraction per library = 1.04%). Library amplification efficiency was robust with each library prep method and efficiency was uniform across all 96 unique index primer combinations. Each bar represents the average of at least 2 technical replicates.

 

 

Figure 5: Unique Dual Index Primer Pairs allow identification and discarding of index-hopped reads.

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One set of the 96 NEBNext Ultra II FS DNA libraries (Figure 2) was used to assess the accuracy of read classification and filtering of barcode-hopped reads using unique dual indices. The 96 libraries were pooled at equimolar concentration and the pool was sequenced on both the NovaSeq 6000 (2x150 bp) and MiSeq® (2 x 76 bp) instruments. Reads were demultiplexed using the Picard ExtractIlluminaBarcodes tools considering both indices, and classified into the following categories:

 

-Demultiplexed reads: expected i5 and i7 barcode combination

-Identified index hopping: expected barcodes, but not in expected combination

-Dark clusters: N or G reads (not observed on MiSeq due to 4-color chemistry)

-Phi X: any reads matching the universal primer sequence (not present in MiSeq experiment)

-Other: any remaining reads not fitting into the above categories


Demultiplexing with a single i5 or i7 index (or a non-unique combination of i5 and i7) incorrectly assigns ~5% of reads on the NovaSeq and 0.6% of reads on the MiSeq, leading to misassignment of reads between samples and problems for downstream analyses. Unique dual indices successfully identify and enable rejection of reads of unknown provenance during demultiplexing.

アダプターオリゴと併用可能なライブラリー調製試薬

NEBNext Ultra II DNA
NEBNext Ultra II (Directional) RNA

キット内容

・NEBNext Adaptor for Illumina

・USER Enzyme

・10 µM each NEBNext 96 Unique Dual Index Primer Pairs Plate (Set 2) *1 *2

 

*1. 96 穴プレートとして提供、各ウェルにユニークなプライマーペア(プレミックス)が含まれています。

*2. インデックス配列はマニュアルもしくはサンプルシートをご覧ください。