NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2) (E6442)

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NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2)

NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2)

NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2)カタログ番号:E6442

カタログ番号	サイズ	濃度	価格	保存温度
E6442S	96 rxns		~~¥77,500~~ ¥58,120	-20C
E6442L	384 rxns		~~¥278,000~~ ¥208,500	-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

備考

備考：新製品 ( 2020年1月20日 )

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 #E6440 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

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

16 libraries were prepared with 100 ng of Human NA19240 genomic DNA (Coriell Institute), using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805). 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), NEB #E6440. 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 Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2) cluster evenly on the Illumina NovaSeq™ 6000

96 libraries produced using the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805) and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2) were pooled at equimolar concentrations and sequenced on the Illumina MiSeq® and NovaSeq® 6000 instruments. 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 on both platforms.

Figure 4: NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Set 2) amplify libraries with equal efficiency

Human NA19240 genomic DNA (Coriell Institute) was used to prepare 96 libraries using either the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805), Covaris-sheared DNA with the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), the NEBNext Enzymatic Methyl-seq (EM-seq) Kit (NEB #E7120), or the NEBNext Ultra II DNA Library Prep Kit combined with bisulfite conversion. Universal Human Reference RNA was used to prepare 96 libraries using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB #E7760). Libraries were PCR-amplified using the NEBNext Multiplex Oligos (96 Unique Dual Index Primer Pairs Set 2) 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.