NEBNext Sample Preparation Technologies
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NEBNext ® Sample Preparation Technologies FOR NEXT GENERATION SEQUENCING GENOMIC DNA LIBRARY PREPARATION PRODUCT NEB # RECOMMENDED INPUT AMOUNTS NEBNext Ultra ™ II FS DNA Library Kit for Illumina/ NEBNext Ultra II FS DNA Library Prep with Sample Purification Beads E7805, E6177 100 pg – 0.5 µg DNA NEBNext Ultra II DNA Library Prep Kit for Illumina/ NEBNext Ultra II DNA Library Prep with Sample Purification Beads E7645, E7103 500 pg – 1 µg DNA NEBNext Oligos (12-, 96-plex and dual index primers, including unique pairs) E7335, E7500, E7710, E7730, E6609, E7600, E7780, E6440 ILLUMINA ® - COMPATIBLE SAMPLE PREP SOLUTIONS RNA LIBRARY PREPARATION PRODUCT NEB # RECOMMENDED INPUT AMOUNTS NEBNext Ultra II Directional RNA Library Prep Kit for Illumina/ NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads E7760, E7765 5 ng – 1 µg Total RNA (rRNA Depletion Workflow) NEBNext Ultra II RNA Library Prep Kit for Illumina/ NEBNext Ultra II RNA Library Prep with Sample Purification Beads E7770, E7775 10 ng – 1 µg Total RNA (poly(A) mRNA workflow) NEBNext Oligos (12-, 96-plex and dual index primers, including unique pairs) E7335, E7500, E7710, E7730, E6609, E7600, E7780, E6440 NEBNext Poly(A) mRNA Magnetic Isolation Module E7490 ULTRA II RNA WORKFLOW • Barcodes incorporated using NEBNext primers • Unique dual-, dual-, and single-barcode primer options available NEBNext Oligos • Fragmentation by incubation with divalent cations (e.g., Mg ++ ) or enzymes (e.g., RNase III) • Hybridization of random primers • Reverse transcriptase lacking RNase H activity is optimal (does not degrade RNA in RNA:DNA complex) • For directional RNA library preparation, Actinomycin D is added: – To inhibit DNA-dependent DNA Polymerase activity of RT & inhibit second strand synthesis/increase strand specificity • Generation of nicks & gaps in RNA by RNase H, enabling second strand synthesis by nick translation • Sealing of breaks in second strand by E. coli DNA ligase • For Directional RNA library preparation, second strand labeled with uracils by dUTP incorporation • Generation of blunt, phosphorylated ends • Addition of single A 3´ overhang (enables ligation to adaptors with single T overhangs) • Ligation of short adaptors (contain sequences required downstream) • NEBNext adaptors increase ligation efficiency & minimize adaptor-dimer formation RNA Fragmentation & Random Priming First Strand cDNA Synthesis Second Strand cDNA Synthesis End Repair, dA-Tailing & Adaptor Ligation 5´ 3´ U A U U U U T 3´ 5´ U A T 5´ 3´ 5´ 3´ U U U U 5´ 3´ U A T 3´ 5´ U A T 5´ 3´ 3´ 5´ NNNNN 5´ 3´ m7G NNNNN NNNNN NNNNN 5´ 3´ AAAAA 5´ 3´ 5´ 3´ DIRECTIONAL NON-DIRECTIONAL • Removal of uracils in NEBNext Adaptor loop by USER Enzyme (to make accessible for PCR) Directional Only • Selective removal of second strand through excision of uracils by USER Enzyme • Result is single-stranded molecule with different adaptor-derived sequences on each end • Amplification using a high-fidelity polymerase: – Selects for molecules with an adaptor at each end – Increases library yield – Incorporates barcodes/ indices to enable multiplexing, and P5 & P7 sequences required downstream PCR Enrichment U Excision 5´ 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ P7 BC 5´ P5 5´ 3´ USER 5´ 5´ 3´ 3´ 5´ 5´ P7 BC P7 P5 P5 BC 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ USER be INSPIRED drive DISCOVERY stay GENUINE ULTRA II DNA WORKFLOW ULTRA II FS DNA WORKFLOW • Enzymatic fragmentation • Generation of blunt-ended fragments (filling in/ chewing back 3´ & 5´ overhangs) • 5´ phosphorylation • Creation of single 3´ A overhang enables ligation to adaptors with single T overhangs DNA Fragmentation, End Repair & dA-Tailing 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 5´ A A A A A 3´ A • Fragmentation by acoustic shearing, nebulization or enzyme-based methods DNA Fragmentation (Not Required for ChIP) 3´ 5´ 5´ 3´ 5´ 3´ 3´ 3´ 5´ 5´ 3´ 5´ • Barcodes incorporated using NEBNext primers • Single- or dual-barcode primer options available, including unique dual barcodes NEBNext Oligos • Amplification using a high-fidelity polymerase: – Selects for molecules with an adaptor at each end – Increases library yield – Incorporates barcodes/indices to enable multiplexing, and P5 & P7 sequences required downstream PCR Enrichment 5´ 5´ 3´ 3´ 5´ 5´ P7 BC P7 BC 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ P5 P5 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ P5 BC2 BC2 P5 BC1 P7 BC1 P7 BC1 P7 BC1 P7 SINGLE BARCODE DUAL BARCODES • Ligation of short adaptors (contain sequences required downstream) • A novel hairpin loop structure increases ligation efficiency & minimizes adaptor-dimer formation Adaptor Ligation 5´ 3´ U A T 3´ 5´ U A T • Generation of blunt-ended fragments (filling in/ chewing back 3´ & 5´ overhangs) • 5´ phosphorylation • Creation of single 3´ A overhang enables ligation to adaptors with single T overhangs End Repair & dA-Tailing 5´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 5´ A A A A A 3´ A • Removal of uracils in NEBNext Adaptor loop by USER ® Enzyme (to make accessible for PCR) U Excision 5´ 3´ 5´ 3´ USER NEBNEXT DIRECT ® TARGET ENRICHMENT This unique approach enriches a wide range of genomic targets directly from DNA, eliminating the need to generate a library prior to enrichment. Enriched regions are converted to a sequencer-ready library through simple enzymatic manipulations that remove off-target sequence and ligate universal adaptors. The short single-bait design enables flexible targeting and compatibility with degraded clinical samples, while incorporation of UMIs enables superior determination of allelic frequencies. • Genomic DNA is fragmented, either mechanically or using included nicking reagents Fragmentation • Biotin bait targets both strands (shown for one strand) Denaturation & bait hybridization • Enzymatic removal of off-target sequence 3´ blunting of DNA • Creation of single 3´ A overhang enables ligation to adaptor with a single T overhang dA-tailing • 3´ hairpin loop adaptor is ligated Ligation of 3´ adaptor • Bait is extended to the 5´ end of the randomly sheared fragment, creating a variable 5´ end of the target read 5´ blunting of DNA Ligation of 5´ UMI adaptor • 5´ Unique Molecule Index (UMI) addition enables removal of PCR duplicate reads Adaptor cleaving • 3´ adaptor is cleaved PCR amplification • Sample index is added during PCR PRODUCT NEB # NEBNext Direct Custom Ready Panels E6631 NEBNext Direct Cancer HotSpot Panel E7000 NEBNext Direct BRCA1/BRCA2 Panel E6627 Target region Genomic DNA 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ Biotin bait 3´ 5´ 5´ 3´ Streptavidin bead 3´ 5´ 5´ 3´ 5´ 3´ 3´ 5´ A T 3´ adaptor 3´ 5´ A T A 3´ 5´ 5´ UMI adaptor T A 3´ 5´ T 3´ 5´ 5´ 3´ A Sequencing-ready fragment 3´ 5´ 5´ 3´ Incorporated sample index T A NEBNEXT RNA DEPLETION Efficient removal of ribosomal RNA (rRNA) from total RNA for human, mouse and rat samples. This method works well for both low-quality/degraded RNA (including FFPE RNA) and high-quality, intact RNA. • Removal of abundant RNAs (e.g., > 80% of total RNAs are rRNAs) or enrichment of mRNAs • NEBNext Library Prep kits are compatible with either method RNA Enrichment (RNA Depletion or Poly(A) mRNA Isolation) 5´ m7G 3´ AAAAA rRNA • Total RNA contains greater than 80% rRNA (red) Total RNA ssDNA probes • Single-stranded DNA probes hybridize specifically to rRNA molecules Binding of ssDNA probes RNase H • RNase H degrades the hybridized RNA (rRNA) rRNA degradation by Ribonuclease H (RNase H) DNase I • DNase I degrades the DNA probes Probe degradation by DNase I & clean up • Non-rRNA species (blue) are enriched rRNA-depleted RNA PRODUCT NEB # NEBNext rRNA Depletion Kit (Human/Mouse/Rat)/ NEBNext rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads E6310, E6350 NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat)/ NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads E7750, E7755 NEBNEXT LIBRARY QUANT KIT Accurate quantitation of NGS libraries is essential for maximizing sequencing data output and quality. qPCR is considered to be the most accurate and effective method of library quantitation, providing considerably higher consistency and reproducibility than electrophoresis or spectrophotometry, which measure total nucleic acid concentration. Amplification-based methods quantitate only those molecules that contain both adaptor sequences, thereby providing a more accurate estimate of the concentration of library molecules that can be sequenced. Library Quantitation Workflow Typical results from the NEBNext Library Quant Kit with 4 standards on a Bio-Rad ® CFX96 Touch ™ , with default settings. Amplification curve (left) and resulting standard curve (right). 4 standards are used to generate the standard curve Standards NTC Library Size adjusted library concentration = 69.8 nM Reagent Preparation Library Dilution Set Up qPCR Data Analysis PRODUCT NEB # NEBNext Library Quant Kit For Illumina E7630 Sodium bisulfite method Converted Sequenced EM-seq method TET2/Oxidation Enhancer APOBEC CCGTCGGACCGC hm m CCGTCGGACCGC CCGTCGGACCGC hm UU GTCGGAUU GC hm m TT GTCGGATT GC TT GTCGGATT GC UU GTCGGAUU GC ca/g ca/g ca/g ca/g m • Protection of 5mC & 5hmC methylation marks with TET2/Oxidation Enhancer • Conversion of non-methylated cytosines with APOBEC • Methylome sequencing with Ultra II DNA (below) PRODUCT NEB # NEBNext Enzymatic Methyl-seq Kit E7120 NEBNEXT ENZYMATIC METHYL-SEQ (EM-seq ™ ) NEW SINGLE CELL/LOW INPUT RNA SMALL RNA WORKFLOW SMALL RNA PRODUCT NEB # RECOMMENDED INPUT AMOUNTS NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1)/ NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 2)/ NEBNext Multiplex Small RNA Library Prep Kit for Illumina (Index Primers 1-48) E7300, E7580, E7560 100 ng – 1 µg Total RNA • Preferential ligation of 5´ adaptor to single-stranded molecules (and therefore not to double- stranded 3´ adaptor:RT primer hybrid molecule) • Result is minimized formation of adaptor-dimers • Hybridization of RT primer to 3´ adaptor- ligated molecules & any remaining 3´ adaptors • Extension from RT primer synthesizes first strand cDNA • Reverse transcriptase lacking RNase H activity is optimal (does not degrade RNA in RNA:DNA complex) 5´ Adaptor Ligation Primer Hybridization First Strand cDNA Synthesis • Input is purified total RNA • Ligation of 5´-adenylated, 3´-blocked, single-stranded DNA adaptor to 3´ end of RNA 3´ Adaptor Ligation 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ App App 3´ 3´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 3´ 3´ App App 5´ 5´ 5´ 5´ 5´ 5´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ App App 5´ 5´ 5´ 5´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ • Amplification with a high-fidelity polymerase: – Selects for molecules with an adaptor at each end – Increases library yield – Incorporates barcodes/indices to enable multiplexing, and P5 & P7 sequences required downstream PCR Enrichment • Ensures that only Small RNAs of interest are included in final library Size Selection 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ 5´ P7 BC 5´ P7 BC 5´ P7 BC 3´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 3´ 3´ P5 P5 P5 Random Primer NN Barcode (BC) P7 Primer NEBNext Adaptor U T DNA Poly(A) Tail AA App 3´ Adaptor 5´ Adaptor RNA Uracil U P5 Primer RT Primer USER Enzyme For more information, visit NEBNext.com V7.0 | 08.19 Printed in the USA on recycled paper (30% PCRF) One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications. ILLUMINA ® is a registered trademark of Illumina, Inc. BIO-RAD ® is a registered trademark of Bio-Rad Laboratories, Inc. CFX96 TOUCH ™ is a trademark of Bio-Rad Laboratories, Inc. © Copyright 2019, New England Biolabs, Inc.; all rights reserved. NEBNEXT FFPE DNA REPAIR MIX Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples significantly damages the nucleic acids within these samples. It can be challenging to obtain high-quality sequence data, especially when sample amounts are limited. The NEBNext FFPE DNA Repair Mix is a cocktail of enzymes optimized and validated for repair of FFPE DNA samples. FFPE DAMAGE TYPE REPAIRED? Deamination of cytosine to uracil Yes Nicks and gaps Yes Oxidized bases Yes Blocked 3´ ends Yes DNA fragmentation No DNA-protein crosslinks No FFPE DNA Sample Preparation Workflow Extract & Purify DNA Shear DNA FFPE DNA Repair Library Construction PRODUCT NEB # NEBNext FFPE DNA Repair Mix M6630 Ability of FFPE DNA damage to be repaired by the NEBNext FFPE DNA Repair Mix PRODUCT NEB # RECOMMENDED INPUT AMOUNTS NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina/ NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module E6420, E6421 Single cells or 2 pg – 200 ng RNA Single cell Total RNA Cell lysis OR mRNA cDNA RT primer 5´ 3´ 5´ 3´ AAAAAA TTTTTT 5´ 3´ 5´ 3´ AAAAAA TTTTTT CCC 5´ 3´ 5´ 3´ AAAAAA TTTTTT CCC GGG GGG Template-switching oligo (TSO) Primer 5´ 3´ 5´ 3´ TTTTTT CCC 5´ 3´ 5´ 3´ TTTTTT CCC 5´ 3´ 5´ 3´ TTTTTT CCC GGG AAAAAA 3´ 5´ 5´ 3´ GGG AAAAAA • Generation of cDNA with a non-templated CCC tail • Template-switching enables production of full-length cDNAs with a common sequence at the 3´ end Template switching • Amplification of the full-length cDNA provides better coverage of 5´ ends cDNA amplification Reverse transcription & non-templated addition • Following a clean-up step, the final cDNA library is ready to move into the Ultra II FS DNA workflow Final cDNA library Final cDNA library 5´ 3´ 5´ 3´ TTTTTT CCC GGG AAAAAA 5´ 3´ 5´ 3´ TTTTTT CCC GGG AAAAAA SINGLE CELL/LOW INPUT RNA WORKFLOW NEW
Transcript of NEBNext Sample Preparation Technologies
NEBNext® Sample Preparation TechnologiesFOR NEXT GENERATION SEQUENCING
GENOMIC DNA LIBRARY PREPARATIONPRODUCT NEB # RECOMMENDED INPUT AMOUNTSNEBNext Ultra™ II FS DNA Library Kit for Illumina/ NEBNext Ultra II FS DNA Library Prep with Sample Purification Beads
E7805, E6177 100 pg – 0.5 µg DNA
NEBNext Ultra II DNA Library Prep Kit for Illumina/ NEBNext Ultra II DNA Library Prep with Sample Purification Beads
E7645, E7103 500 pg – 1 µg DNA
NEBNext Oligos (12-, 96-plex and dual index primers, including unique pairs)E7335, E7500, E7710, E7730, E6609, E7600, E7780, E6440
ILLUMINA®-COMPATIBLE SAMPLE PREP SOLUTIONS
RNA LIBRARY PREPARATIONPRODUCT NEB # RECOMMENDED INPUT AMOUNTSNEBNext Ultra II Directional RNA Library Prep Kit for Illumina/ NEBNext Ultra II Directional RNA Library Prep with Sample Purification Beads
E7760, E7765 5 ng – 1 µg Total RNA (rRNA Depletion Workflow)
NEBNext Ultra II RNA Library Prep Kit for Illumina/ NEBNext Ultra II RNA Library Prep with Sample Purification Beads
E7770, E7775 10 ng – 1 µg Total RNA (poly(A) mRNA workflow)
NEBNext Oligos (12-, 96-plex and dual index primers, including unique pairs)E7335, E7500, E7710, E7730, E6609, E7600, E7780, E6440
NEBNext Poly(A) mRNA Magnetic Isolation Module E7490
ULT
RA
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• Barcodes incorporated using NEBNext primers
• Unique dual-, dual-, and single-barcode primer options available
NEBNext Oligos
• Fragmentation by incubation with divalent cations (e.g., Mg++) or enzymes (e.g., RNase III)
• Hybridization of random primers
• Reverse transcriptase lacking RNase H activity is optimal (does not degrade RNA in RNA:DNA complex)
• For directional RNA library preparation, Actinomycin D is added:– To inhibit DNA-dependent DNA Polymerase activity of RT &
inhibit second strand synthesis/increase strand specificity
• Generation of nicks & gaps in RNA by RNase H, enabling second strand synthesis by nick translation
• Sealing of breaks in second strand by E. coli DNA ligase
• For Directional RNA library preparation, second strand labeled with uracils by dUTP incorporation
• Generation of blunt, phosphorylated ends
• Addition of single A 3´ overhang (enables ligation to adaptors with single T overhangs)
• Ligation of short adaptors (contain sequences required downstream)
• NEBNext adaptors increase ligation efficiency & minimize adaptor-dimer formation
RNA Fragmentation & Random Priming
First Strand cDNA Synthesis
Second Strand cDNA Synthesis
End Repair, dA-Tailing & Adaptor Ligation
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• Removal of uracils in NEBNext Adaptor loop by USER Enzyme (to make accessible for PCR)
Directional Only• Selective removal of
second strand through excision of uracils by USER Enzyme
• Result is single-stranded molecule with different adaptor-derived sequences on each end
• Amplification using a high-fidelity polymerase:– Selects for molecules with
an adaptor at each end– Increases library yield– Incorporates barcodes/
indices to enable multiplexing, and P5 & P7 sequences required downstream
PCR Enrichment
U Excision
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to adaptors with single T overhangs
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• Barcodes incorporated using NEBNext primers
• Single- or dual-barcode primer options available, including unique dual barcodes
NEBNext Oligos
• Amplification using a high-fidelity polymerase:– Selects for molecules with an adaptor
at each end– Increases library yield– Incorporates barcodes/indices to enable
multiplexing, and P5 & P7 sequences required downstream
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• Generation of blunt-ended fragments (filling in/chewing back 3´ & 5´ overhangs)
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NEBNEXT DIRECT® TARGET ENRICHMENT
This unique approach enriches a wide range of genomic targets directly from DNA, eliminating the need to generate a library prior to enrichment. Enriched regions are converted to a sequencer-ready library through simple enzymatic manipulations that remove off-target sequence and ligate universal adaptors. The short single-bait design enables flexible targeting and compatibility with degraded clinical samples, while incorporation of UMIs enables superior determination of allelic frequencies.
• Genomic DNA is fragmented, either mechanically or using included nicking reagents
Fragmentation
• Biotin bait targets both strands (shown for one strand)
Denaturation & bait hybridization
• Enzymatic removal of off-target sequence
3´ blunting of DNA
• Creation of single 3´ A overhang enables ligation to adaptor with a single T overhang
dA-tailing
• 3´ hairpin loop adaptor is ligated
Ligation of 3´ adaptor
• Bait is extended to the 5´ end of the randomly sheared fragment, creating a variable 5´ end of the target read
5´ blunting of DNA
Ligation of 5´ UMI adaptor• 5´ Unique Molecule Index (UMI)
addition enables removal of PCR duplicate reads
Adaptor cleaving• 3´ adaptor is cleaved
PCR amplification• Sample index is added during PCR
PRODUCT NEB #
NEBNext Direct Custom Ready Panels E6631
NEBNext Direct Cancer HotSpot Panel E7000
NEBNext Direct BRCA1/BRCA2 Panel E6627
T
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Target region
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TA
T
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Target region
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T
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Target region
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Target region
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5´3´
Sequencing-ready fragment
3´5´
5´3´
Incorporatedsample index
A
TA
T
T
Target region
GenomicDNA
Biotin bait
Streptavidin bead
3´ adaptor
5´ UMIadaptor
3´5´
5´3´
3´5´
5´
3´ 5´
3´ 5´
3´
5´ 3´
5´ 3´
5´3´
3´5´ A
TA
TA
3´5´ A
3´5´
3´5´
3´5´
5´3´
Sequencing-ready fragment
3´5´
5´3´
Incorporatedsample index
A
TA
NEBNEXT RNA DEPLETIONEfficient removal of ribosomal RNA (rRNA) from total RNA for human, mouse and rat samples. This method works well for both low-quality/degraded RNA (including FFPE RNA) and high-quality, intact RNA.
• Removal of abundant RNAs (e.g., > 80% of total RNAs are rRNAs) or enrichment of mRNAs
• NEBNext Library Prep kits are compatible with either method
RNA Enrichment (RNA Depletion or Poly(A) mRNA Isolation)
5´ m7G 3´AAAAA
rRNA
RNase H
ssDNAprobes
DNase I
• Total RNA contains greater than 80% rRNA (red)
Total RNA
rRNA
RNase H
ssDNAprobes
DNase I
• Single-stranded DNA probes hybridize specifically to rRNA molecules
Binding of ssDNA probes
rRNA
RNase H
ssDNAprobes
DNase I
• RNase H degrades the hybridized RNA (rRNA)
rRNA degradation by Ribonuclease H (RNase H)
rRNA
RNase H
ssDNAprobes
DNase I
• DNase I degrades the DNA probes
Probe degradation by DNase I & clean up
rRNA
RNase H
ssDNAprobes
DNase I
• Non-rRNA species (blue) are enriched
rRNA-depleted RNA
PRODUCT NEB #NEBNext rRNA Depletion Kit (Human/Mouse/Rat)/ NEBNext rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads
E6310, E6350
NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat)/ NEBNext Globin & rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads
E7750, E7755
NEBNEXT LIBRARY QUANT KIT
Accurate quantitation of NGS libraries is essential for maximizing sequencing data output and quality. qPCR is considered to be the most accurate and effective method of library quantitation, providing considerably higher consistency and reproducibility than electrophoresis or spectrophotometry, which measure total nucleic acid concentration. Amplification-based methods quantitate only those molecules that contain both adaptor sequences, thereby providing a more accurate estimate of the concentration of library molecules that can be sequenced.
Library Quantitation Workflow
Typical results from the NEBNext Library Quant Kit with 4 standards on a Bio-Rad® CFX96 Touch™, with default settings. Amplification curve (left) and resulting standard curve (right).
4 standards are used to generate the standard curve
Standards NTC Library
Size adjusted library concentration = 69.8 nM
ReagentPreparation
LibraryDilution Set Up qPCR
DataAnalysis
PRODUCT NEB #
NEBNext Library Quant Kit For Illumina E7630
Sodium bisulfite method
Converted
Sequenced
EM-seq method
TET2/OxidationEnhancer
APOBEC
CCGTCGGACCGC
hm m
CCGTCGGACCGC
CCGTCGGACCGC
hm
UUGTCGGAUUGC
hm m
T T GTCGGAT T GC T T GTCGGAT T GC
UUGTCGGAUUGC
ca/g ca/g
ca/g ca/g
m
• Protection of 5mC & 5hmC methylation marks with TET2/Oxidation Enhancer
• Conversion of non-methylated cytosines with APOBEC• Methylome sequencing with Ultra II DNA (below)
PRODUCT NEB #
NEBNext Enzymatic Methyl-seq Kit E7120
NEBNEXT ENZYMATIC METHYL-SEQ (EM-seq™)NEW
SINGLE CELL/LOW INPUT RNA
SMA
LL RN
A W
OR
KFLO
W
SMALL RNAPRODUCT NEB # RECOMMENDED INPUT AMOUNTS NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 1)/ NEBNext Multiplex Small RNA Library Prep Set for Illumina (Set 2)/ NEBNext Multiplex Small RNA Library Prep Kit for Illumina (Index Primers 1-48)
E7300, E7580, E7560
100 ng – 1 µg Total RNA
• Preferential ligation of 5´ adaptor to single-stranded molecules (and therefore not to double-stranded 3´ adaptor:RT primer hybrid molecule)
• Result is minimized formation of adaptor-dimers
• Hybridization of RT primer to 3´ adaptor-ligated molecules & any remaining 3´ adaptors
• Extension from RT primer synthesizes first strand cDNA
• Reverse transcriptase lacking RNase H activity is optimal (does not degrade RNA in RNA:DNA complex)
5´ Adaptor Ligation
Primer Hybridization
First Strand cDNA Synthesis
• Input is purified total RNA• Ligation of 5´-adenylated, 3´-blocked,
single-stranded DNA adaptor to 3´ end of RNA
3´ Adaptor Ligation
5´ 3´ 5´ 3´
5´ 3´5´
5´
3´
3´App
App
3´
3´5´
5´
3´
3´3´
5´
5´
3´
3´App
App
5´
5´
5´
5´
5´
5´
5´
5´
3´
5´ 3´5´ 3´
5´
5´
3´
3´App
App5´
5´
5´5´
5´
5´
3´5´
3´5´
3´5´
5´3´
5´3´
5´3´
• Amplification with a high-fidelity polymerase:– Selects for molecules with an adaptor at each end– Increases library yield– Incorporates barcodes/indices to enable
multiplexing, and P5 & P7 sequences required downstream
PCR Enrichment
• Ensures that only Small RNAs of interest are included in final library
Size Selection 5´ 3´5´3´
5´ 3´5´3´
5´5´
5´
5´3´
5´3´
5´3´
3´
5´
5´
5´ 3´3´
5´P7BC
5´P7BC
5´P7BC
3´
5´
5´
5´ 3´3´
3´
5´
5´
5´3´ 3´
3´
5´
5´
5´ 3´3´
3´
5´
5´
5´3´ 3´
3´
5´
5´
5´ 3´3´
3´
5´
5´
5´3´ 3´
P5
P5
P5
Random Primer
NN Barcode (BC) P7 Primer
NEBNextAdaptor
U TDNA Poly(A) TailAA App 3´ Adaptor 5´ Adaptor
RNA UracilU P5 Primer RT Primer
USER Enzyme
3´
3´5´
5´
3´
3´3´
5´
5´
3´
3´App
App
5´
5´
5´
5´
5´
5´
5´
5´
For more information, visit NEBNext.com
V7.0 | 08.19
Printed in the USA on recycled paper (30% PCRF)
One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.
ILLUMINA® is a registered trademark of Illumina, Inc. BIO-RAD® is a registered trademark of Bio-Rad Laboratories, Inc. CFX96 TOUCH™ is a trademark of Bio-Rad Laboratories, Inc.
© Copyright 2019, New England Biolabs, Inc.; all rights reserved.
NEBNEXT FFPE DNA REPAIR MIX
Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples significantly damages the nucleic acids within these samples. It can be challenging to obtain high-quality sequence data, especially when sample amounts are limited. The NEBNext FFPE DNA Repair Mix is a cocktail of enzymes optimized and validated for repair of FFPE DNA samples.
FFPE DAMAGE TYPE REPAIRED?Deamination of cytosine to uracil Yes
Nicks and gaps Yes
Oxidized bases Yes
Blocked 3´ ends Yes
DNA fragmentation No
DNA-protein crosslinks No
FFPE DNA Sample Preparation Workflow
Extract &Purify DNA
ShearDNA
FFPEDNA Repair
LibraryConstruction
PRODUCT NEB #
NEBNext FFPE DNA Repair Mix M6630
Ability of FFPE DNA damage to be repaired by the NEBNext FFPE DNA Repair Mix
PRODUCT NEB # RECOMMENDED INPUT AMOUNTS
NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina/ NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module
E6420, E6421 Single cells or 2 pg – 200 ng RNA
Final cDNA library
Single cell Total RNA
Cell lysis
mRNA
cDNA
Primer
RT primer
5´ 3´5´3´
AAAAAATTTTTT
5´ 3´5´3´
AAAAAATTTTTT
5´
CCC
3´5´3´
AAAAAATTTTTTCCC
GGG
GGGTemplate-switchingoligo (TSO)
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
3´ 5´5´ 3´GGG AAAAAA
OR
Final cDNA library
Single cell Total RNA
Cell lysis
mRNA
cDNA
Primer
RT primer
5´ 3´5´3´
AAAAAATTTTTT
5´ 3´5´3´
AAAAAATTTTTT
5´
CCC
3´5´3´
AAAAAATTTTTTCCC
GGG
GGGTemplate-switchingoligo (TSO)
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
3´ 5´5´ 3´GGG AAAAAA
OR
Final cDNA library
Single cell Total RNA
Cell lysis
mRNA
cDNA
Primer
RT primer
5´ 3´5´3´
AAAAAATTTTTT
5´ 3´5´3´
AAAAAATTTTTT
5´
CCC
3´5´3´
AAAAAATTTTTTCCC
GGG
GGGTemplate-switchingoligo (TSO)
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
3´ 5´5´ 3´GGG AAAAAA
OR
Final cDNA library
Single cell Total RNA
Cell lysis
mRNA
cDNA
Primer
RT primer
5´ 3´5´3´
AAAAAATTTTTT
5´ 3´5´3´
AAAAAATTTTTT
5´
CCC
3´5´3´
AAAAAATTTTTTCCC
GGG
GGGTemplate-switchingoligo (TSO)
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
3´ 5´5´ 3´GGG AAAAAA
OR
• Generation of cDNA with a non-templated CCC tail
• Template-switching enables production of full-length cDNAs with a common sequence at the 3´ end
Template switching
• Amplification of the full-length cDNA provides better coverage of 5´ ends
cDNA amplification
Reverse transcription & non-templated addition
• Following a clean-up step, the final cDNA library is ready to move into the Ultra II FS DNA workflow
Final cDNA library
Final cDNA library
Single cell Total RNA
Cell lysis
mRNA
cDNA
Primer
RT primer
5´ 3´5´3´
AAAAAATTTTTT
5´ 3´5´3´
AAAAAATTTTTT
5´
CCC
3´5´3´
AAAAAATTTTTTCCC
GGG
GGGTemplate-switchingoligo (TSO)
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
5´ 3´5´3´ TTTTTTCCC
GGG AAAAAA
3´ 5´5´ 3´GGG AAAAAA
OR
SING
LE CELL/LO
W IN
PU
T R
NA
WO
RK
FLOW
NEW
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