PicoPLEX™ DNA-seq Kit Instruction Manual
Contents
1. DNA segq Kit is that it provides robust and reproducible library construction for NGS with a single cell Washing cultured cells Minimize non cellular DNA contaminations by washing cells with sterile nuclease free 1X PBS buffer free of Mg Cat ul in the cell lysis step of the reaction and BSA freshly prepared from a 10X PBS stock The carryover PBS volume must not exceed 2 5 gt Cells obtained by approaches described above can be stored for future use at 80 C by flash freezing or processed directly following the PicoPLEX DNA seq Protocol Positive and Negative Controls Include appropriate positive and negative controls in the experimental design to help verify that the test reactions proceeded as expected A good choice for the positive reference control is single donor gDNA Always prepare fresh dilutions of gDNA to use as the positive control Include a negative control No Template Control NTC without cells or gDNA containing only 2 5 uL of PBS or TE buffer 10 mM Tris pH 8 0 0 1 mM EDTA The reference DNA positive control and experimental samples cells should work equally well If the experimental samples contain any carryover contaminant s in the buffer the downstream reactions may be impacted inclusion of controls would help explain such problems Please refer to the Appendix 1 for details on preparing working dilutions of the reference gDNA from stock solutions Dual Index Plate PicoPLEX D2. Master Mix to each well 5 Add the indexes to the PCR plate or tube s mM Using clean pipet tips pierce the seal above the target index of the Dual Index Plate discard the tip s used for piercing Use a clean pipet tip to collect 5 uL of a specific index and add to the reaction Gently mix the contents several times with a pipettor Final reaction volume at this stage will be 50 uL Amplification Reaction Volume Reaction From Pre Amplification Reaction 15 uL Amplification Master Mix Index Oligonucleotide Note Follow the index plate handling instructions Appendix 2 to avoid cross contamination 6 Seal the PCR plate or tubes tightly and centrifuge briefly to collect the contents to the bottom of each well Note Use optical sealing tape if a real time thermal cycler is used 7 Return the plate or tube s to the real time thermal cycler with the heated lid on and perform the Amplification Reaction using the cycling conditions from the table below Amplification Reaction Step Temperature Time Number of cycles Step 1 95 C 4 min 1 cycle 95 C 20 sec Step 2 63 C 25 sec A cycles 72 C AO sec 95 C 20 sec 72 C 55 sec Cae Step 3 Step 4 4 C Hold Acquire fluorescence data at this step if monitoring amplification in real time QAM 106 002 I 8 At the end of library amplification remove the PCR plate or tube s from the thermal cycler and centrifuge briefly to collect
3. tateuaeeens I3 Library Quanti catio Nenen a a a A A AEETI TETO 14 SEQUENCING INECOMMENGALIONS cecoteecceecteectes hese ctestees n bnon RER ADE a Ei teens n A nE I5 Analysis or the Sequencing Reads consranniinntin iran iT ETTE A N A N EEVT I5 Appendix l Reference DNA Dilution cessssssessssensscesescsscesscesesccesescseesesceneaceeeeesceeeseeeeesecseees 16 Appendix 2 Dual Index Plate signee ee een eaten enhanc T ET 16 OV EIVICW saute cate taaa dadn a auna i aaa aaa oeoo ao aoaaa 16 Handling for Low Throughput Applications ccscccssssscscssssesecesessssescssssessscesessssessscesesescesesceeeeseceeeaseeeeeees 17 Product Description PicoPLEX DNA seq Kit Cat R300381 48 reactions is intended for amplifying genomic DNA from single cells to reliably detect chromosomal aneuploidies and copy number variations CNV on Illumina Next Generation Sequencing NGS platforms Applications include pre implantation genetic screening PGS using blastomeres and trophectoderm cells and CNV detection in circulating tumor cells CTCs where single cell amplifications are required For more information visit www rubicongenomics com products PicoPLEX DNA seq Kit Contents Table 1 PicoPLEX DNA seq Kit contents Cap Color Name Shipping and Storage PicoPLEX DNA seq Kit Cat R300381 is shipped on dry ice The kit should be stored at 20 C upon arrival Quality Control PicoPLEX DNA seq Kit is tested against predefined speci
4. the contents to the bottom of each well At this stage samples can be processed for library purification immediately or stored frozen at 20 C for later processing PicoPLEX DNA seg Library Processing for Illumina Next Generation Sequencing Overview This section contains guidelines for processing PicoPLEX DNA seq libraries for Illumina NGS In some cases recommended protocols are listed Library Purification by AMPure XP beads while in others general guidelines are given For more information please contact our technical support staff at support rubicongenomics com Libraries prepared from each sample will contain the sample specific index combinations that were selected at the time of the amplification An equal volume of each individual library containing their unique index combinations are pooled into one tube for further processing This library pool is purified to remove unincorporated primers and other reagents Once purified the library is quantified prior to NGS The library pooling process purification protocol quantification protocols and recommendations for sequencing are described in the following sections of the PicoPLEX DNA seq protocol Pool Prepared Libraries Purify Pooled Library Quantify Pooled Library Next Generation Sequencing NGS Figure 5 Workflow for processing the PicoPLEX DNA seq amplified libraries for Illumina NGS Library Pooling for Purification Pool
5. 702 7 7 D705 D706 2 7 8 9 10 11 12 114 OOOQOQOOO0000O0 OO QOOOOO0000O OO O0O000000 OWO OO000000 SGO ODQO00000 O OQO00000 OOOOQOO000O0 8 OOOOOOOOO000O0 Le D503 C D D504 D505 m D506 A G Index i5 D501 D502 D503 D504 D505 D506 D507 D508 Sequence TATAGCCT ATAGAGGC COTAT er GGCTCTGA ee eemme TAATCTTA enceeem GTACTGAC Index i7 D701 D702 D703 D704 D705 D706 Sequence ATTACTCG TCCGGAGA SECICATT GAGATTCC ATTCAGAA GAATTCGT Figure 7 Dual index plate map with well locations Single use index plate representing well locations left of 48 dual indexes with appropriate i7 columns and i5 rows index combinations the sequences of respective indexes are shown in the table right Wells A7 H12 are blank wells and do not contain any index oligonucleotides QAM 106 002 Product Use Limitations PicoPLEX DNA seq Kit is intended for Research Use Only It may not be used for any other purposes including but not limited to use in diagnostics forensics therapeutics or in humans PicoPLEX DNA seq may not be transferred to third parties resold modified for resale or used to manufacture commercial products without prior written approval of Rubicon Genomics Inc This product is protected by US Patent 8 206 913 and pending The index sequences correspond to Illumina Index sequences for multiplexing and are copyrighted to Illumina Inc O
6. NA seq kit is designed for high throughput applications and includes a 96 well single use Dual Index Plate containing 48 unique dual indexes Each of 48 wells contains a combination of i5 and i7 indexes prepared with Illumina 8nt sequences to avoid potential sample misidentifications due to barcode switching Figure 4 Each well contains sufficient volume of the dual index pair for a single use The plate is sealed with QAM 106 002 6 foil that can be pierced with a pipet tip to collect the required amount of the dual index pair to assemble the reactions It is recommended to design your experiment to use the entire plate of barcodes to avoid contamination problems However the Dual Index Plate can also be used for low level multiplexing of a small number of samples The plate should not be used more than 4 times If all 48 index pairs are not used at the same time it is important to seal the opened wells with laboratory labeling tape to avoid cross contamination Please refer to the index plate handling instructions in Appendix 2 It is also very important to select appropriate dual index combinations such that they are unique and meet Illumina recommended compatibility requirements Please refer to Illumina s technical manuals TruSeq Sample Preparation Pooling Guide Illumina Part 15042173 Rev A 2013 for additional information Barcode combinations may also be evaluated using Illumina Experiment Manager Software for compatibi
7. TV JANG RUBICON GENOMICS PicoPLEX DNA seq Instruction Manual Single Cell Library Preparation tor Ilumina NGS Platforms Contents Product Description oroni Seta Sascha estes Beveled taeda este od R Deaeded itd eatin wath A Kit Contents oroi ects et see EE as dad astern unbasdewslcg i cashaus hast NTO Shipping ahd Storage seats tetas aces chee ee eie ac ceca tect ta ce seceetcacaclecc ata clea caches a cicleace thoes cctalee sac cea teeltacadeaets COTA CONTO cscs seca sca ct satan cence sonacecactacaeae E Sale by MMT Vasco neers cheat ca haha ce cei cea atnicaee cet cette ce ata ca ua et cc aheeneenat acetate aes kechnhical Assistante enr eae a aa e aa e aE eE EEE e eE e Introduction to Single Cell Next Generation Sequencing s ssesesossssesesssresesessoresesessosesessssesesessese Introduction to the PicoPLEX DNA seq Kit csssssessscesesssseecsceeesssceeesceeesesceeeseeeeesceeesseeeeeees ONEVIEW nT TE T T E E T E cect cee ene tee Br MUNG UI IO ec a a a a a a PicoP LEX DNA S69 WOrRIOW ticcri E E E ciclds cs sects sce te ea ctcl te Gettin Startede Additional supplies and equipment needed sscsssssssssssssssssssssrsssssssssnssssncessacsesceesssenssseasersacsesecenssceass Thermal Cycler Considerations snoniieiienn nnan E EEEE AAO NEEE Thermal cycling and heated lid Monitoring amplification during the Library Amplification Step Selecting PCR Plates tubes Starine hd Fo Rene nr ere ee en A A e
8. Yellow 0 2 UL 5 To each 5 uL of equilibrated sample from steps 1 3 above containing a cell s b reference gDNA or c NTC s add 5 uL of Cell Extraction Master Mix to assemble the Lysis Reactions as shown in the table below Do not touch the cell or DNA sample with the pipet tip Final reaction volume at this stage will be 10 uL Lysis Reaction Volume Reaction Sample 5 UL Cell Extraction Master Mix 5 uL Total Volume 10 uL 6 Seal the PCR plate using an appropriate sealing film or close the tube s tightly Centrifuge briefly to ensure the entire volume of the reaction is collected at the bottom of each well QAM 106 002 8 Place the plate or tube s in a thermal cycler with heated lid on and perform the Lysis Reaction using the protocol in the table below Lysis Reaction Incubation Conditions Temperature Time At the end of the Lysis Reaction after the cycler reaches 22 C remove the plate or tube s and centrifuge briefly Proceed to the Pre Amplification step Note Following the cell lysis step continue pre amplification in the same plate or tube s Pre Amplification Step Pre Amplification Reagents Pre Amplification Reagents Reagent Cap Color Pre Amp Buffer Red Pre Amp Enzyme White Pre Amplification Protocol Prepare a Pre Amplification Master Mix on ice as described in the table below for the chosen number of reactions mix gently several times and keep on ice unt
9. aliquot of this diluted sample onto a Bioanalyzer high sensitivity DNA chip Agilent Technologies Inc cat 5067 4626 QAM 106 002 14 150 300 500 1000 FO bp 150 300 500 1000 7000 bp Figure 6 Bioanalyzer analysis of libraries prepared using PicoPLEX DNA seq Libraries were prepared using 15 pg of reference DNA or NTC with PicoPLEX DNA seq Kit An aliquot of each library was diluted at 1 15 in TE buffer and 1uL of this diluted sample was loaded on Bioanalyzer using a high sensitivity DNA chip Agilent Technologies Inc Electropherogram results Panels A and B showing a broad size range distribution M Markers 1 Library prepared using 15 pg reference gDNA 2 NTC Sequencing Recommendations PicoPLEX DNA seq Kit generates libraries which are ready for cluster amplification and sequencing on the Illumina NGS platforms using standard Illumina reagents and protocols for multiplexed libraries Libraries prepared using PicoPLEX DNA seq Kit result in a broad size distribution of library fragments Figure 6 typically ranging from 300 to 1000 bp total size 200 to 900 bp insert size It is important to note that when libraries consist of a broad range of fragments the clustering process preferentially amplifies shorter fragments as the longer fragments tend to cluster less efficiently To achieve optimal cluster density on the Illumina flow cell it is important to adjust the DNA concentration used for clustering based on these
10. aration of Illumina compatible NGS libraries from single cells PicoPLEX DNA seq libraries is used to characterize unique genomic signatures that include chromosomal aneuploidies and copy number variations in PGS in Vitro Fertilization IVF clinics as well as in cancer research with single tumor cells e g CTCs Introduction to the PicoPLEX DNA seq Kit Overview The PicoPLEX DNA seq Kit is designed to generate Illumina NGS ready libraries in a single tube from a single cell in less than 2 5 hrs The kit includes all necessary reagents for extracting DNA from a single cell and preparing amplified NGS ready libraries Dual indexes barcodes included in the kit avoid potential sample misidentifications due to barcode switching The resulting library is ready for Illumina NGS instruments using standard Illumina sequencing reagents and protocols PicoPLEX DNA seq Kit offers robust and reproducible amplification of DNA from a single cell for limited coverage sequencing analysis and is not intended for high coverage deep sequencing such as de novo sequencing and or whole genome sequencing Principle PicoPLEX DNA seq is based on Rubicon Genomics patented PicoPLEX technology for single cell genomic DNA gDNA amplification which uses multiple cycles of quasi random priming for reproducible library construction Figure 1 PicoPLEX DNA seq Kit follows the same three step workflow of the PicoPLEX WGA Kit In the first step a single cel
11. fications using Next Generation Sequencing NGS to ensure product quality and consistency Safety Information Follow standard laboratory safety procedures and wear a suitable lab coat protective goggles and disposable gloves to ensure personal safety as well as to limit potential cross contaminations during the sample preparation and subsequent amplification reactions For more information please refer to appropriate Material Safety Data Sheets MSDS available online at www rubicongenomics com Technical Assistance For technical support with any of the Rubicon Genomics Inc products please contact Rubicon s technical support team by email support rubicongenomics com or call at 734 677 4845 Monday thru Friday from 9 AM till 5 PM EST QAM 106 002 Introduction to Single Cell Next Generation Sequencing Next Generation Sequencing NGS enables precise and impartial analysis of the genome for many applications including detecting chromosomal aneuploidies copy number variations CNV insertions and deletions These are especially relevant in Pre implantation Genetic Screening PGS in IVF clinics and cancer genetics In each of these applications the starting material is limited due to the use of single cells and requires reproducible amplification of the genome as a prerequisite for the downstream analysis by NGS PicoPLEX DNA seq Kit has been developed specifically to meet this growing need and facilitate prep
12. hannel pipet tip to collect the required amount of the dual index pair to assemble the reactions It is recommended that your experiment be designed to use the entire plate of barcodes to avoid contamination problems However the Dual Index Plate can also be used for low level multiplexing of a small number of samples The plate should not be used more than 4 times If all 48 index pairs are not used at the same time it is important to seal the opened wells with a laboratory labeling tape to avoid cross contamination It is very important to select appropriate dual index combinations such that they are unique and meet Ilumina recommended compatibility requirements Please refer to Illumina s technical manuals TruSeq Sample Preparation Pooling Guide Illumina Part 15042173 Rev A 2013 for additional information Barcode combinations may also be evaluated using Illumina Experiment Manager Software for compatibility QAM 106 002 16 Handling for Low Throughput Applications If the entire Dual Index Plate is not used it is critical to follow the instructions below to avoid cross contamination 1 After removing indexes of choice cover any pierced or used index wells with scientific tape such as VWR General Scientific Tape 0 5 Cat 89097 920 2 Thoroughly wipe the seal with 70 ethanol and allow it to dry completely 3 Replace the plastic lid return the Dual Index Plate to its sleeve and store at 20 C D701 D
13. he instructions to ensure that there is no barcode cross contamination If the entire Dual Index Plate will not be used please refer to Appendix 2 for handling procedures No more than 4 freeze thaw cycles are recommended for the Dual Index Plate Amplification Protocol 1 a If monitoring in real time Prepare 20X EvaGreen Fluorescein dye mix or dye recommended for your thermal cycler mM Mix 90 ul of 20X EvaGreen dye Cat 31000 T 20X EvaGreen dye in water Biotium Inc mixed with 10 uL of 1 500 dilution of Fluorescein Fluorescein Calibration Dye Catalog 170 8780 Bio Rad Laboratories M Use 2 5 uL of this mix per reaction b If not monitoring in real time Replace dyes with water in the Master Mix Step 3 2 Prepare the Dual Index Plate E Thaw the Dual Index Plate for ten minutes on the bench top E Spin the Dual Index Plate in a table top centrifuge to ensure its contents are at the bottom of the well a Thoroughly wipe the foil seal with 70 ethanol and allow it to dry 3 Prepare the Amplification Master Mix as described in the table below for the chosen number of reactions mix gently several times and keep on ice until used QAM 106 002 10 Amplification Master Mix Component Cap Color Volume Reaction Amplification Buffer Amplification Enzyme Fluorescent Dyes Step 1 a Nuclease Free Water Total 30 UL Reaction 4 Remove the seal on the PCR plate or open the tube s and add 30 uL of the Amplification
14. iginal stock DNA 2 Pipet 14 uL of TE buffer low EDTA into a microcentrifuge tube and add 6 uL of the 1000 pg uL reference DNA working stock solution from step 1 to achieve a final concentration of 300 pg uL 3 Pipet 36 uL of TE buffer low EDTA into a second microcentrifuge tube and add 4 uL of the 300 pg uL DNA stock solution from step 2 to achieve a final concentration of 30 pg uL 4 Pipet 18 uL of TE buffer low EDTA into a third microcentrifuge tube and add 6 uL of 30 pg uL stock solution from step 2 to achieve a final concentration of 7 5 pg uL 5 To prepare the final concentrations E 60 pg of reference DNA input Use 2 uL of the 30 pg L DNA from step 3 E 15 pg of reference DNA input Use 2 uL of the 7 5 pg uL DNA from step 4 Appendix 2 Dual Index Plate Overview PicoPLEX DNA segq Kit is designed for high throughput applications and includes a 96 well single use Dual Index Plate containing 48 different dual indexes Each of 48 wells contains a combination of i5 and i7 indexes prepared with Illumina 8nt sequences to avoid potential sample misidentifications due to barcode switching Figure 7 The Illumina dual index oligonucleotides contained in this kit cannot be substituted with index oligonucleotides from any other sources The plate is intended for high throughput applications each well contains sufficient volume of the dual index pair for a single use The plate is sealed with foil that can be pierced with a multic
15. il used Pre Amplification Master Mix Component Cap Color Volume Reaction Pre Amp Buffer Red 4 8 UL Pre Amp Enzyme White 0 2 UL Remove the seal on the plate or open the tube s and to each lysis reaction mixture add 5 uL of Pre Amplification Master Mix to assemble the Pre Amplifications as shown in the table below Final reaction volume at this stage is 15 uL Pre Amplification Reaction Volume Reaction From Lysis Reaction 10 uL Pre Amplification Master Mix 5 uL Total Volume 15 pL Seal the plate or tube s tightly and centrifuge briefly to collect the contents to the bottom of each well Return the plate or tube s to the thermal cycler with the heated lid on and perform the Pre Amplification Reaction using the cycling conditions in the table below QAM 106 002 9 Pre Amplification Reaction Step Temperature Time Number of cycles Step 1 95 C 2 min 1 cycle Step 3 4 C 5 After the cycler reaches 4 C remove the plate centrifuge briefly and continue to the Library Amplification Step Note Following Pre Amplification step continue the amplification reaction in the same plate or tube s maintained at 4 RCA Library Amplification Step Amplification Reagents Amplification Master Mix Reagent Cap Color Amplification Buffer Orange Amplification Enzyme Blue Nuclease Free Water Clear Fluorescent Dyes Dual Index Plate Note It is critical to handle the Dual Index Plate following t
16. l or up to 10 cells are efficiently lysed to release gDNA Note that DNA may also be used in this step In the second step proprietary quasi random primers bind to selective sites on the gDNA and through a linear amplification create a highly reproducible library In the third step the library is further amplified exponentially with primers containing unique dual barcodes suitable for Illumina NGS QAM 106 002 2 Single Cell J Step 1 Cell Lysis Accumulating n Po VF 2 ad Step 2 Preamp 12 cycles Extend Figure 1 PicoPLEX DNA seg technology A three step single tube reaction that starts with a single cell Cellular g DNA extracted in step 1 is used as template for multiple cycles of quasi random priming and linear amplification followed by exponential library amplification PicoPLEX DNA seq Workflow The PicoPLEX DNA seq Kit workflow is highly streamlined Figure 2 and consists of the following three modules E Cell Lysis Module for efficient lysis and release of g NA E Pre Amplification Module for reproducible and consistent priming and multiple cycles of linear amplification of the released DNA E Amplification Module for exponential amplification and Illumina compatible dual indexing for NGS The three step PicoPLEX DNA seq workflow takes place in the same tube or plate and is completed in less than 3 hours QAM 106 002 3 Single cells in PCR plate Prepare Enzyme Extraction Dispense Mas
17. ligonucleotide sequences 2007 2012 Illumina Inc All rights reserved BioAnalyzer is a registered trademark of Agilent Technologies Inc Agencourt AMPure XP isa registered trademark of Beckman Coulter Inc EvaGreen is a registered trademark of Biotium Inc LabChip is a registered trademark of Caliper Life Sciences Inc TruSeq is a registered trademark of Illumina Inc Qubit is a registered trademarks of Life Technologies QIAquick are registered trademarks of Qiagen NanoDrop is a trademark of Thermo Fisher Scientific Inc HOA RUBICON GENOMICS 4355 Varsity Drive Suite E Ann Arbor MI 48108 T 1 734 677 4845 F 1 734 477 9902
18. lity Note The Illumina dual index oligonucleotides contained in this kit cannot be substituted with index oligonucleotides from any other sources Note Avoid repeated freezing and thawing of the plate No more than 4 freeze thaw cycles are recommended Index i5 Sequence Index i7 Sequence D501 TATAGCCT D701 ATTACTCG D502 ATAGAGGL D702 TUCGGAGA D503 COCTATCCT D703 COCTCATT D504 SGCTCTGA D704 GAGATTCC D505 AGEGCLAAGL D705 ATTCAGAA D506 TAATCT TA D706 GAATTCOT D507 CAGGACGT D508 SGTACTGAL Figure 4 Dual index plate map with well locations Single use index plate representing well locations left of 48 dual indexes with appropriate i7 columns and i5 rows index combinations the sequences of respective indexes are shown in the table right Wells A7 H12 are empty Preparation of Master Mixes It is recommended to prepare a master mix with appropriate buffers and enzymes at each step based on the number of reactions to be performed Transfer the enzymes to ice just prior to use and centrifuge briefly to ensure all the contents are at the bottom of the tube Thaw the buffers vortex briefly and centrifuge prior to use Keep all the components and master mixes on ice Once the master mix is prepared mix the contents several times gently with a pipettor while avoiding introduction of excessive air bubbles and briefly centrifuge prior to dispensing into the PCR plate QAM 106 002 7 PicoPLEX DNA seg Library Prepa
19. m temperature In the meantime prepare fresh 80 ethanol enough for steps 4 and 6 below 1 Re suspend the AMPure XP reagent by gentle vortexing until no visible pellet is present at the bottom of the container 2 Ina 1 5 mL tube mix 100 uL of AMPure XP reagent with a 100 uL aliquot of the pooled library ensuring a 1 1 v v ratio Gently mix by pipette 10 times to achieve a homogeneous solution and incubate the tube at room temperature for 5 min 3 Pulse spin the sample s on a bench top centrifuge and place the tube in a magnetic stand Wait for 2 min or until the beads are completely bound to the side of the tube s and the solution is clear 4 With the tube s in the magnetic stand and without disturbing the pellet use a pipette to aspirate off and discard the supernatant Add 300 uL of freshly prepared 80 ethanol to the pellet 5 With the tube s in the magnetic stand rotate each tube 90 degrees and wait until all the beads come to a halt DO NOT INVERT TUBE RACK Repeat this step three more times 6 With the tube s in the magnetic stand and without disturbing the pellet use a pipette to aspirate off and discard the supernatant Add 300 uL of freshly prepared 80 ethanol to the pellet 7 With the tube s in the magnetic stand and without disturbing the pellet turn each tube 90 degrees and wait until all the beads come to a halt DO NOT INVERT TUBE RACK Repeat this step three more times 8 With the tube s i
20. n the magnetic stand and without disturbing the pellet use a pipette to aspirate off and discard the supernatant 9 Pulse spin the sample s using a low speed bench top centrifuge place into a magnetic stand and wait for 2 min or until the beads are completely bound to the side of the tube s 10 With the tube s in the magnetic stand and without disturbing the pellet use a pipette to aspirate off and discard any residual ethanol without disturbing the pellet Leaving the cap open incubate the sample s in a heating block at 37 C for 2 3 min or until the pellet is dry DO NOT OVER DRY THE PELLET S 12 Elute the DNA by re suspending the beads with 50 uL of 1x TE buffer pH 8 0 13 Pulse spin the sample s using a low speed bench top centrifuge and place it into a magnetic stand and let the beads bind to the side of the tube s completely for 2 min until the solution is clear 14 While keeping the sample s in the magnetic stand without disturbing the pellet transfer the supernatant with a pipette into a new tube If not used immediately the purified library can be stored at 20 C QAM 106 002 3 Library Quantification There are several approaches available for library quantification including real time PCR UV absorption fluorescence detection and sizing and quantification using the Agilent Bioanalyzer It is important to understand the benefits and limitations of each approach Real
21. or plates that are compatible with the thermal cyclers and or real time thermal cyclers used Use appropriate caps or sealing films and seal thoroughly to eliminate evaporation during cycling conditions Evaporation could reduce robustness and reproducibility of the reactions Starting Material Cells Single mammalian cells 1 10 cells from a broad range of sources can be used Examples of cells for reliably detecting chromosomal aneuploidies by NGS include blastomeres and trophectoderms CTCs cultured cells clonally expanded cells micro manipulated single cells and flow sorted single cells QAM 106 002 5 Genomic DNA In place of whole cells small amounts less than 6 pg to 60 pg of purified genomic DNA can be used as starting material for library preparation Purified eukaryotic prokaryotic fungal or viral DNA can also be used as starting material Key Considerations for Cell Preparation Cell collection Single cells collected by dilution micro manipulation and flow sorted stained by surface antibodies or unstained are suitable with the kit Cell fixation should be avoided for optimal results Clonally expanded cells Use of clonally expanded cells with genetic homogeneity will help achieve optimal results as many cultured cell lines have unstable genomes not evident when averaging analysis even over a few cells Number of cells Up to 10 cells can be used per reaction however the major advantage of the PicoPLEX
22. preferences With libraries made from PicoPLEX DNA seq loading 16 pM of a library with an average size of 300 bp is a good starting point for calculating the amount to use with the Illumina MiSeq v3 It is very important to add at least 5 Phix DNA to the library prior to loading on the flow cell to achieve necessary diversity Analysis of the Sequencing Reads The first 11 cycles of each read will contain quasi random bases introduced during the PicoPLEX DNA seq library preparation For sequence alignment either trim the initial 14 bases from each read or begin calibration and data collection at base position 15 QAM 106 002 I5 Appendix Reference DNA Dilution Single donor human genomic DNA is ideal for use as positive control DNA e g catf GH 180M Human Genomic DNA male 1 mg mL cat GH 180F Human Genomic DNA female 1 mg mL from Zyagen or cat DD7101 control DNA Male 2800M 10 pg mL from Promega Follow the steps below to prepare the working dilutions for the reference genomic DNA At the end of each dilution step mix the contents gently and centrifuge briefly before going to the next dilution step Always use freshly diluted DNA for positive control reactions All reference DNA dilutions are carried out using low EDTA TE buffer pH 8 0 10 mM Tris pH 8 0 0 1mM EDTA in 500 uL low binding microcentrifuge tubes 1 Prepare a working stock solution of 1000 pg uL by appropriately diluting a 2 3 uL aliquot of the or
23. ration Protocol Cell Lysis Step Cell Lysis Step Reagents Cell Lysis Step Reagents Reagent Cap color Cell Extraction Buffer Green Extraction Enzyme Dilution Buffer Violet Cell Extraction Enzyme Yellow Note Assemble all reactions in thin wall 96 well PCR plates or tube s that are compatible with the thermal cycler and or the real time cycler used Cell Lysis Protocol 1 Test samples Equilibrate cells 1 to 10 or DNA 6 60 pg to a final volume of 5 uL by adding an appropriate amount of Cell Extraction Buffer CEB Note Ifa single cell is isolated in PBS do not exceed 2 5 uL of PBS adjust the final volume to 5 uL with CEB 2 Positive control reaction using reference DNA Assemble reactions using freshly diluted reference gDNA at an input amount of 15 pg refer to Appendix 1 for preparing dilutions of reference DNA by adding 2 uL of a 7 5 pg uL dilution Add 3 uL of CEB to each tube to bring the final volume of each reaction to 5 ul 3 Negative control reactions No Template Controls NTCs Assemble NTC with 2 5 uL of PBS or TE buffer 10 mM Tris pH 8 0 0 1 mM EDTA and adjust the final volume to 5 uL with CEB 4 Prepare Cell Extraction Master Mix as described in the table below for the chosen number of reactions mix gently several times and keep on ice until used Cell Extraction Master Mix Component Cap color Volume Reaction Extraction Enzyme Dilution Buffer Violet 4 8 UL Cell Extraction Enzyme
24. re OE ONAA Geno rne NA eee nce ene tore Umar eo ear tr A E CP Rte ae PEO on eet ee Key Considerations for Cell PreparatioMesenirennriori i nna E eens Cell collection Clonally expanded cells Number of cells Washing cultured cells Positive and Negative ControlSisenriir nonnen n AA AN ATO T Dual INGOX Plate rant cence the atte ame aaa a aaa e Ea eE TEn N GA Preparation Or Master MIKES eniinn annia nN TO E E PicoPLEX DNA seq Library Preparation Protocol s esessseesesossesesesessesesessssesesessosesesessesesesessesesessese CEI EY SIS SCC Dicnirenn aee A AIA NATET TET ETOO Collis ysis Stop RCAgeIts agase ii nan S E eeesiadisess 8 CUE AE EE E I E T E A EE E E T E 8 PRrE AmiplifiCatlOMn Step sn tetecatetteceteteete eee eee eee eee ene eee eee eens 9 PresAimplincation Reagents arnor E a baad diceriarenteisatuide 9 Pre Api aon PrO Olesea EE E N E E vata iaumene A NEA 9 Library AMmpiication Step earsnin on nst an N NOOR 10 PIMP aao R EA S aa EE A mania N A man awalua tenons 10 Amp ined on PV OLOCO Viscan ace E eesti Naesdaneaideen ERN 10 PicoPLEX DNA seq Library Processing for Illumina Next Generation Sequencing 12 OVERVIEW eceran na niente ieee iene nineteen eee 12 Library Purification by AMPure XP beads ssssssssssssssesesseessesessosscesersossccenscsacsceseceacsceseesacscesensacsceserses 12 AMPure XP PrOtocOlnt lt sccttca tte igeesens tice tite isataduenteatsaaseeesans ARA send aasnarsade tear
25. sing a real time thermal cycler with the addition of fluorescent dyes not provided with the kit to the reaction Figure 3 If a regular thermal cycler is used instead there is no need to add the dyes substitute an appropriate amount of nuclease free water to adjust the volumes in the Amplification QAM 106 002 4 Master Mix In the absence of real time monitoring library amplification can be analyzed by gel or by analysis of an aliquot of the library using the Agilent Bioanalyzer see Library Quantification page 15 Depending on the real time instrument used select an appropriate calibration dye and mix with EvaGreen detection dye mix see Amplification Protocol Step 1 For some real time instruments calibration dye may not be needed please refer to the real time thermal cycler instrument s user manual Amplification Curves 15 pg Reference DNA a SSN N M N mampll gt L N v Q U Q N o i O 3 Ll v 2 pu U ce Amplification cycles Figure 3 Real time analysis of library amplification using PicoPLEX DNA seq A typical real time amplification analysis of libraries prepared with PicoPLEX DNA seq Kit using three single H929 cells blue or three reference DNA samples 15 pg red relative to NTC grey Results obtained using CFX96 Touch Real Time PCR Detection System with EvaGreen and fluorescein as the dyes Selecting PCR Plates tubes Select appropriate tubes
26. ter Mix Perform Cell Lysis Reaction Prepare Pre Amplification Dispense Master Mix Perform Pre Amplification Reaction Prepare Dispense Appropriate Dispense Master Mix Index to Each Well Dual Index Plate Perform Amplification Reaction AMPure XP Cleanup Next Generation Sequencing NGS Figure2 PicoPLEX DNA seq sequencing workflow overview Steps involved in PicoPLEX library preparation starting from a single cell for Ilumina NGS Note that tubes may replace the PCR plate Getting Started Additional supplies and equipment needed Thermal cycler real time instrument recommended centrifuge 96 well thin wall PCR plates or PCR tubes PCR plate seals low binding barrier tips phosphate buffered saline 1X PBS Mg free Ca free and BSA free 80 ethanol single donor reference DNA positive control AMPure XP beads nuclease free water Optional fluorescent dyes EvaGreen Biotium Cat 31000 T and Fluorescein Calibration Dye Bio Rad Laboratories Catalog 170 8780 recommended only needed if monitoring amplification in real time Thermal Cycler Considerations Thermal cycling and heated lid Use a thermal cycler equipped with a heated lid that can handle 50 uL reaction volumes Set the temperature of the heated lid to100 105 C to avoid sample evaporation during incubation and cycling Monitoring amplification during the Library Amplification Step Amplification can be monitored u
27. the samples by combining equal volume aliquots of each PicoPLEX DNA seq library each containing a unique dual barcode combination Typically a 10 uL aliquot from each library is adequate and the remainder of the library can be stored at 20 C The total volume obtained at the end of pooling will vary depending on the number of libraries pooled For example if 12 libraries are pooled then the final volume of the pool is 120 uL if 48 libraries are pooled then the volume is 480 uL A 100 uL aliquot of this pooled library is sufficient for AMPure XP purification purposes Library Purification by AMPure XP beads AMPure XP is the preferred method of library purification due to its ability to preserve the sequence complexity Do not use QIAquick cleanup or other silica based filters for purification as this will result in incomplete removal of primers The ratio of AMPure XP beads to library DNA will determine the size selection characteristics of the library The ratio is also application dependent For most NGS based CNV detection purposes a good starting point is mixing the beads and the sample s at a 1 1 ratio For more information please refer to the vendor s recommendations on AMPure XP protocols for DNA purification QAM 106 002 12 Library purification reagents supplied by the user Library Purification Reagents AMPure XP Protocol Note It is important to bring all the samples and reagents to be used to roo
28. time PCR based approaches such as Illumina library quantification kit from KAPA Biosystems quantify the library molecules that carry the Illumina adapter sequences on both ends and therefore reflect the quantity of the clustering competent library molecules This approach assumes a relatively uniform size of sheared or fragmented starting gDNA inserts used for library construction On the other hand UV absorption fluorescence detection based methods i e Nanodrop Thermo Scientific Qubit 2 0 Fluorometer Life Technologies Inc or Quant iT PicoGreen dsDNA Assay Kit Life Technologies Inc simply quantify total nucleic acid concentration These methods do not discriminate adapter presence an indication of clustering competence and offer no information about the size of the library molecules The Agilent Bioanalyzer system provides sizing and quantitation information about the library analyzed but not about the clustering competency To quantify PicoPLEX DNA seg libraries by real time qPCR select the appropriate instrument specific Illumina library quantification kit from KAPA Biosystems Pooled purified libraries are diluted 50 000 to 500 000 fold and used as the template for quantification It is recommended to use 300 bp as the size for calculating the library concentration To quantify PicoPLEX DNA seq libraries by using the BioAnalzyer Figure 6 remove an aliquot of each library and dilute 1 15 Load a 1uL
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