Archer™ Universal DNA Reagent Kit v2 for Illumina®
Contents
1. Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction and each tube contains a different Illumina Index 1 Barcode Primer 4 2 1 lf you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Be sure to track which barcodes were used to ensure barcode compatibility when used in later experiments Centrifuge briefly to ensure lyophilized material is in the bottom of the tube To the Second PCR tube on ice add Purified library DNA Step 3 16 18 uL Liquid GSP2 Mix 2 uL Total 20 uL Allow the pellet to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube Incubate the reaction as specified in the panel specific Instructions for Use found at http www archerdx com documents variantplex instructions Note the ramp rate and consult your instrument user s manual to confirm that this setting Is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to gt 100 C Post Second PCR AMPure XP Beads Purification 4 7 4 8 4 9 4 10 4 L1 4 12 4 13 4 14 4 15 4 16 Add 16 uL of AMPure XP beads to the 20 uL reaction for a ratio of 0 8X Make sure that AMPure XP Beads are fully resuspende2. Instructions for Use LF WAX po ss rA 4 y aig OE a Lag y m Z d ARCHER Archer Universal DNA Reagent Kit v2 for Illumina AKO037 8 Table of Contents Archer Universal DNA Reagent Kit v2 for IIUMINa cessccscessccccecsccscsecscccssececesssscecessuceesesesceseesseteseseeceesen 1 MOV SO os OVC UC cosas ecco gece cesses ant ee coco cece ae mcs E eons A AEE E A E EAS 1 Be ISON O estat sta ceet E E E E E E 2 POOU E DE Or O ee E a E a E 2 NT NSS a0 I ONO TA E T EEE EA E EEA A A A EEE A E 2 W a6 CO O E A E E E E E EER 3 KIE CONTON reee a AE E E EE EE E T E 3 Materials Required But Not SUPPICC nce esc sssscsseceecscccescecesescecescscsecusecescsceressecesescenescscesescesesceseresceceeecerececesees 3 General Precautions c cccccccccceseeccesecccesseccsessesesesceecssesescsccccssesenescscescssenessesescecesescecesestenesseseressecessccenseceeeseecenessenetesees 4 SOA SING Ir EIn e INNS EEEE EE EE ENEE NN NEEE AE EAEN AAA 4 Sample Multiplexing aos resistence ner smunaec dew esonsticesinnrsna wien njanwtownedieos ci mane vestps or sieti pune bu cist asi twat cetivagteeebeal gee sabi awdatinanseenseaeet 4 Barcode TIVO I ac ce ace toga EAE EEEE EE AE 4 Input Nucleic Acid Concentration and WIC WON sepaccxsctsaacaacenseceectencnceascacaectnaceneateaccueciesssestesacesegleaccuadeeccuatuauedecstaten D Bo ODE eE EE E E E S 5 Removal of Divalent Cations and EDTA from Input Nucleic
3. assays and Molecular Barcode MBC Adapters allows users to construct Illumina MiSeg or NextSeq ready libraries from total nucleic acid or DNA samples Archer DNA Assays Universal Reagent Kit Molecular Barcode MBC Adapters Gene specific primers that Platform specific kits that Platform specific half functional enable amplification of your contain all of the reagents universal adapters for specific genes of interest needed for library preparation advanced analysis For Research Use Only Not for use in diagnostic procedures 2 Archer Universal DNA Reagent Kit v2 for IIlumina IFU AK0037 8 Rev B ARCHER i Workflow Overview 50 uL of DNA H 0 Vv Fragmentation End Repair dA Tailing MBC Adapters 2 y Adapter Ligation w iE AA DE vY SEs WW Second PCR Quantify Library and Sequence Kit Contents 500 mM Tris HCl pH 8 0 SAQ020 Ultra Pure Water SA0021 Ultra Pure Water for Ethanol Dilution SA0022 Lyophilized Reagents a Step 1 Fragmentation End Repair dA Tailing SAQ039 b Step 2 Adapter Ligation SA0005 c Step 3 First PCR SA0109 d Step 4 Second PCR SA0110 a Materials Required But Not Supplied 1 Archer MBC Adapters for Illumina Agencourt AMPure XP Beads Cat A63881 Life Technologies DynaMag Cat 12331D 100 ethanol ACS grade KAPA Biosystems Library Quantification Kit IIlumina Universal Cat KK4824 ae ce 3 Archer Univer
4. 0 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 2 12 After the final wash briefly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With the caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 2 13 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 24 uL of 10 mM Tris HCl 2 14 Place the AMPure bead solution back on magnet for 2 minutes 2 15 Carefully transfer 22 uL of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 3 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C Step 3 First PCR NOTE The Archer Universal Reagent Kits do not contain gene specific primers GSPs in the reaction pellet GSP1 mix is a part of the Archer VariantPlex Panel Reference the panel instructions for use prior to beginning this section as each panel may have unique cycling conditions 3 1 Gently open the First PCR SA0109 foil pouch by tearing along the indents located at the top of the silver package 3 2 Remove the clear 8 tube strip Each tube in the strip provides a single reaction 3 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the rema
5. 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 1 1 8 After the final wash briefly spin down and carefully remove remaining supernatant while taking Care not to disturb the beads With caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 1 1 9 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 55 uL 10mM Tris HCl 1 1 10 Place the AMPure bead solution back on magnet for 2 minutes 1 1 11 Carefully transfer 53 uL of the clean DNA solution to a fresh 200 uL PCR tube Be sure to avoid transferring beads to the fresh tube Ensure that your starting input is still 100 200ng total nucleic acid or 50 100ng DNA You may reserve 2 uL of the eluate to measure the concentration or quality of the input material Protocol step 1 Fragmentation End Repair dA Tailing 12 1 3 1 4 L5 1 6 1 7 1 8 6 Enter the following program into a thermal cycler and start the program Be certain to set the instrument s heated lid set to 2100 C When the thermal cycler block reaches 4 C pause the program Step Incubation Temperature Incubation Time 1 ANG 1 min 2 37 C 12 min 3 VIAL 20 min 4 4 C Hold Gently open the Fragmentation End Repair dA Tailing SA0039 foil pouch by tearing along the indents located at th
6. Acid qu ccc cece cccteenetescccessssesescccesseseseeeseeseecsesceceresees s POCO E E ee eee ee a eee ee 6 Step 1 Fragmentation End Repair GAT QUT csc ce ete ey aren ete ete oe aoe een ncen eave 6 T AT E a 9 ee eee ce ne Tn Ym TY EY TY TP TT I eT Sy OO 1 a te ee ee Le re cer eee ne nee ene een ee eee ee EE ee 8 oy Ac Oi at CIO 608 ore E ee EE ee Tere en eee eee re eee Sree eee een ver emer en ee ere eer 9 Step 9 Quantify Library and SOQUCNCE cccccccccccesccccsnneccseosssnsssercsecscnssecnansssensesessusssetanessersssesensenedssneseseserensesesenenensecerens 11 For more information please visit http WWW arCherdX COM c ccccsccscessscssessesesssescesssesessscssessscsseseeseceseacensseseenesesees 12 1 Archer Universal DNA Reagent Kit v2 for Illumina IFU AK0037 8 Rev B ARCHER SS Revision History From Rev To Rev Change Deleted PCR1 and PCR2 cycling conditions refer to the panel specific Instructions for Use Deleted Miseg and NextSeg loading recommendations refer to the panel specific Instructions for Use Product Description The Archer Universal DNA Reagent Kit v2 for Illumina can be used in conjunction with Archer DNA assays utilizing the power of next generation sequencing to improve mutation detection The kit contains reagents necessary to perform library preparation on various sample types Modular Assay Format The Archer Universal DNA Reagent Kit v2 for Illumina used in conjunction with Archer
7. C in the sealed foil pouch with the provided desiccant 2 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 2 4 Transfer 50 uL of the Fragmented End Repaired dA tailed DNA sample with the annealed Illumina MBC Adapters Step 113 into the tube containing Adapter Ligation mix Allow pellet to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 2 5 Incubate the reaction as follows If a thermal cycler is used either set the thermal cycler lid to off or leave it open during the incubation 7 Archer Universal DNA Reagent Kit v2 for IIlumina IFU AKQ037 8 Rev B ARCHER Incubation Step Incubation Temperature Time 1 16 C 30 min 2 22 C 30 min Post Ligation AMPure XP Beads Purification 2 6 Add 40 uL of AMPure XP beads to the 50 ul reaction for a ratio of 0 8X Make sure that AMPure XP Beads are fully resuspended by vortexing prior to adding to the sample 2 7 Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture 2 8 Incubate for 5 minutes at room temperature 2 9 Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear 2 10 Carefully pipette off and discard supernatant without disturbing the beads 2 11 Without disturbing the separated beads wash by adding 200 ul of 70 ethanol while on the magnet and incubate for 3
8. d by vortexing prior to adding to the sample Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture Incubate for 5 minutes at room temperature Place beads on magnet for 2 4 minutes or until beads are pelleted and solution is clear Carefully pipette off and discard supernatant without disturbing the beads Without disturbing the separated beads wash by adding 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes After the final wash briefly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 24 uL of 10 mM Tris HCl Place the AMPure bead solution back on magnet for 2 minutes Carefully transfer 22 uL of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 5 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C 10 Archer Universal DNA Reagent Kit v2 for Illumina IFU AKO037 8 Rev B ARCHER step 5 Quantify Library and Sequence 5 1 Use the KAPA Biosystems gPCR kit KK4824 for Illumina to quantify the conce
9. e top of the silver package Remove the blue 8 tube strip Each tube in the strip provides a single reaction 1 4 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Centrifuge briefly to ensure lyophilized material is in the bottom of the tube Place the DNA Fragmentation End Repair dA Tailing reaction tubes on ice Transfer 50 uL of the DNA from step 1 1 11 if cleanup was necessary to the Fragmentation End Repair dA Tailing reaction tubes Gently resuspend the lyophilized pellet by pipetting up and down 4 or b times while keeping the tubes on ice then centrifuge briefly Place DNA Fragmentation End Repair dA Tailing tubes into block of paused thermal cycler at 4 C and resume the program Archer Universal DNA Reagent Kit v2 for Illumina IFU AKO037 8 Rev B ARCHER 1 9 When the thermal cycling program is complete gently open the pouch labeled Archer MBC Adapters for Illumina by tearing along the indents located at the top of the silver package Keep the samples in the chilled thermal cycler or on ice until they are ready for transfer 110 Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction and each tube contains a different Illumina MBC Adapter For example reactions 1 through 8 corre
10. eads 3 12 Without disturbing the separated beads wash by adding 200 uL of 70 ethanol while on the magnet and incubate for 30 sec at room temperature Carefully remove ethanol and discard Repeat this procedure for a total of 2 washes 3 13 After the final wash briefly spin down and carefully remove remaining supernatant while taking care not to disturb the beads With caps open dry beads at room temperature for 5 minutes Take care not to over dry the beads as this can reduce yield 3 14 Remove tubes from the magnet and elute DNA by thoroughly resuspending the beads in 24 uL of 10 mM Tris HCl 3 15 Place the AMPure bead solution back on magnet for 2 minutes 3 16 Carefully transfer 22 ul of the purified solution to a fresh 200 uL PCR tube or proceed directly to Step 4 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C step 4 Second PCR NOTE The Archer Universal Reagent Kits do not contain gene specific primers GSPs in the reaction pellet GSP2 mix is a part of the Archer VariantPlex Panel Reference the panel instructions for use prior to beginning this section as each panel may have unique cycling conditions 4 1 Gently open the Second PCR SA0110 foil pouch by tearing along the indents located at the top of the Silver package 9 Archer Universal DNA Reagent Kit v2 for Illumina IFU AKO037 8 Rev B ARCHER 4 2 4 3 44 4 5 4 6
11. equest 2015 ArcherDX Inc All rights reserved Archer and Archer VariantPlex are trademarks of ArcherDX Inc Illumina NextSeg and MiSeg are registered trademarks of Illumina Inc Agencourt AMPure and FormaPure are registered trademarks of Agencourt Biosciences Corporation a Beckman Coulter company Life Technologies and DynaMag are trademarks of Thermo Fisher Scientific Inc KAPA Biosystems is a registered trademark of KAPA Biosystems Inc RNase Away is a registered trademark of Molecular Bio Products Inc For more information please visit http www archerdx com ArcherDX Inc lis 2477 55 Street Suite 202 A R C E EY Boulder CO 80301 303 357 9001 PA www archerdx com 12 Archer Universal DNA Reagent Kit v2 for Illumina IFU AK0037 8 Rev B
12. ining unused tubes in the sealed foil pouch at 4 C in the sealed foil pouch with the provided desiccant 3 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 34 To the First PCR tube on ice add 8 Archer Universal DNA Reagent Kit v2 for Illumina IFU AKO037 8 Rev B ARCHER Purified library DNA Step 2 15 18 pL Liquid GSP1 Mix 2 pL Total 20 4L 3 5 Allow the pellet to dissolve and then gently pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 3 6 Incubate the reaction as specified in the panel specific Instructions for Use found at http www archerdx com documents variantplex instructions Note the ramp rate and consult your instrument user s manual to confirm that this setting is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to gt 100 C Post First PCR AMPure XP Beads Purification 3 7 Add 16 uL of AMPure XP beads to the 20 uL reaction for a ratio of 0 8X Make sure that AMPure XP Beads are fully resuspended by vortexing prior to adding to the sample 3 8 Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture 3 9 Incubate for 5 minutes at room temperature 3 10 Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear 3 11 Carefully pipette off and discard supernatant without disturbing the b
13. lose the bottle cap to minimize evaporation Removal of Divalent Cations and EDTA from Input Nucleic Acid Note If your input DNA or total nucleic acid is already free of divalent cations and EDTA skip to step 1 2 1 1 Purify DNA or total nucleic acid with AMPure XP beads Note that some total nucleic acid or DNA will be lost during purification so provide 2x the amount needed for library preparation LLE LL dh lice 1 1 4 EMSA 1 1 6 If the DNA or total nucleic acid is in a volume of less than 50 uL adjust the volume to 50 uL with Ultra Pure Water If the volume is greater than 50 uL scale the volume of AMPure XP beads appropriately see step 1 1 2 Add 90 uL of AMPure beads to the reaction for a ratio of 1 8X If the DNA or total nucleic acid is in a volume greater than 50 uL scale the volume of AMPure up such that the ratio of AMPure to DNA ratio is 1 8X Make sure that AMPure XP Beads are fully resuspended by vortexing prior to adding the sample Vortex well or pipette 10 times to mix and visually inspect the color of the sample to ensure a homogenous mixture Incubate for 5 minutes at room temperature Place tubes on magnet for 2 4 minutes or until beads are pelleted and solution is clear Carefully pipette off and discard supernatant without disturbing the beads D Archer Universal DNA Reagent Kit v2 for Illumina IFU AKO037 8 Rev B ARCHER 1 1 7 Without disturbing the separated beads wash by adding
14. mina Index 1 Primers the run may fail due to low barcode diversity In this example it is best to use eight different Archer MBC Adapters paired with eight different Illumina Index 1 Primers Input Nucleic Acid Concentration and Purification e Recommended Input Start with 100 ng 200 ng of total nucleic acid input or 50 ng 100 ng of purified DNA e Be sure that DNA is in buffers that do not contain EDTA i e use Tris HCI and not Tris EDTA buffer e For DNA or total nucleic acid stored in buffers containing EDTA an initial buffer exchange with Agencourt AMPure XP Beads is required before DNA fragmentation Because total nucleic acid from Agencourt FormaPure extraction of FFPE sources is eluted in EDTA free buffer you may proceed directly to step 1 2 and skip AMPure XP cleanup of your sample In step 1 7 add 100 ng 200 ng of total nucleic acid in a total volume of 50 uL If you are using a kit other than FormaPure carefully check the contents of the final storage buffer to determine if it contains EDTA or divalent cations Before You Begin e Make fresh 10 mM Tris HCl o Mix 30 uL 500 mM Tris HCI pH 8 0 SA0020 with 1470 uL Ultra Pure Water SA0021 e Makefresh 70 ethanol o Add 14 mL 100 ethanol ACS grade not included to entire bottle containing Ultra Pure Water for Ethanol Dilution SAQ022 o Note the date on which ethanol is added 70 ethanol is appropriate for use for one week after mixing When not in use tightly c
15. ntration of each library Assume a 250 bp fragment length After quantification pool the barcoded libraries at equimolar concentrations and sequence on an Illumina Miseq or NextSeg Refer to the appropriate Archer VariantPlex Panel s instructions for use to determine the maximum number of samples that can be sequenced together If you are unfamiliar with qPCR MiSeg or NextSeq please reference the appropriate protocols 5 2 Run the Miseg or NextSeq using the read level sequence in the table below In addition a reference sample sheet is available for download at http www archerdx com mbc adapters Fill out the sample Sheet according to the appropriate Illumina protocol Level Read Length R1 Read 1 151 R2 Index Read 1 8 R3 Index Read 2 8 R4 Read 2 151 5 3 Load MiSeg or NextSeg according to recommendations in the panel specific Instructions for Use found at found at http www archerdx com documents variantplex instructions 5 4 Upon completion of the run the data should be analyzed using Archer Analysis at http www archerdx com analysis Limitations of Use For Research Use Only Not for use in diagnostic procedures 11 Archer Universal DNA Reagent Kit v2 for IIlumina IFU AKQ037 8 Rev B ARCHER ere ac rd a gt This product was developed manufactured and sold for in vitro use only The product is not suitable for administration to humans or animals SDS sheets relevant to this product are available upon r
16. or NextSeg multiple samples should be sequenced simultaneously Samples can be identified through a unique nucleotide sequence that is part of the molecular barcode adapter ligated to the nucleic acid sequence of interest during library construction and which is subsequently read during the sequencing process The unique nucleotide sequence is often termed an index The Archer Universal DNA Reagent Kit v2 for Illumina utilizes a combination of two indices to distinguish between samples The first index is added just before Step 2 Adapter Ligation and is embedded in the Archer MBC Adapters for Illumina The second index is added in Step 4 Second PCR and is embedded in Illumina Index 1 Primers within the PCR2 lyophilized pellets n order to maintain appropriate coverage depth it is recommended that users determine the maximum number of samples that can be run on a MiSeg or NextSeg flow cell In general larger panels with more targets will reguire higher sequencing coverage depth and should be run with fewer samples Refer to the applicable VariantPlex Panel instructions for use for multiplexing recommendations Barcode Diversity The Illumina MiSeq and NextSeg will work best when index diversity within a run is high For example if eight Samples are included in a run and the user chooses to use only one MBC Adapter paired with eight different 4 Archer Universal DNA Reagent Kit v2 for IIlumina IFU AKQ037 8 Rev B ARCHER Illu
17. sal DNA Reagent Kit v2 for IIlumina IFU AK0037 8 Rev B ARCHER 6 Archer VariantPlex Panel 7 If nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure Catt A33342 for extraction 8 Illumina MiSeg Reagent Kit v2 or v3 9 NextSeg 500 Mid Output v2 300 cycle Cat FC 404 2003 10 NextSeg 500 High Output v2 300 cycle Cat FC 404 2004 General Precautions e Read the entire protocol before beginning e lake note of stopping points where samples can be frozen at 20 C and plan your workflow accordingly e Use good laboratory practices to minimize cross contamination of nucleic acid products e Always use PCR tubes microfuge tubes and pipette tips that are certified sterile DNase and RNase free e Before starting wipe down work area and pipettes with an RNase and DNA cleaning product such as RNase Away Molecular BioProducts Inc San Diego CA e For consistent library amplification ensure the thermal cycler used in this protocol is in good working order and has been calibrated to within the manufacturer s specifications storage and Handling All components of Archer Universal DNA Reagent Kit v2 for Illumina Part AK0037 8 should be stored between 2 C and 8 C The liquid GSP1 and GSP2 primer tubes should be stored between 10 C and 30 C Allow pouches to warm to room temperature before opening sample Multiplexing In order to efficiently utilize the throughput of the MiSeq
18. spond to MBC Adapters 1 through 8 1 101 CRITICAL Upon removing the 8 tube strip from the pouch position the tubes with the hinges to the back and use a permanent marker to label the tubes 1 through 8 from left to right as shown below Be sure to label and track the index number added to each sample from this point forward 1 10 2 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4 C in the sealed foil pouch with the provided desiccant Be sure to track which indices were used to ensure index compatibility when used in later experiments 1 11 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 1 12 To the Illumina MBC Adapters tube on ice add 50 uL of fragmented DNA Step 1 8 1 13 Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 1 14 Immediately proceed to Step 2 step 2 Adapter Ligation 2 1 Gently open the Adapter Ligation SAO005 foil pouch by tearing along the indents located at the top of the Silver package 2 2 Remove the red 8 tube strip Each tube in the strip provides a single reaction 2 2 1 If you would like to use fewer than 8 reactions carefully detach the appropriate number of tubes using clean scissors or a new razor blade Store the remaining unused tubes at 4
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