KAPA Library Quantification Kits Frequently Asked Questions v2
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1. If the average library fragment size is gt 700 bp e g 454 Titanium Rapid Prep Libraries then increase annealing extension time to 90 sec 9 Can melt curve analysis after qPCR library quantification be used to monitor the quality of the libraries i e detect adaptor dimers average fragment sizes library complexity etc We do not make specific recommendations to perform melt curve analysis after qPCR because it could be very difficult to interpret the results for the following reasons There is a very wide variety of sample types fragment sizes library construction techniques qPCR instruments etc and each of these factors could be expected to affect the melt curve Speaking very generally and superficially the melting temperature profile of DNA fragments above 100 bp tend to be affected more by GC content than by length We therefore think that it would be difficult to infer library fragment size quality from melt curve analysis Each library sample will of course only yield a composite melt curve representing an average across the entire library sample Since even the melt curve profile for a single DNA species can be complex i e multiple local dsDNA to ssDNA transitions within a single fragment even if one could discern multiple peaks it would be difficult to know how to interpret them In fact multiple clearly definable peaks might actually indicate a low quality library because they would imply a low c2. continue to supply these original kits containing the unmodified Primer Premix to existing customers who specifically request to receive the original kit Aside from the Primer Mix all other components of the new kits remain unchanged Existing users may request a sample of the revised Primer Mix for testing New customers and existing customers who do not specifically request the original kit will receive the new kit References for qPCR and NGS library quantification 1 From micrograms to picograms quantitative PCR reduces the material demands of high throughput sequencing Meyer et al Nucleic Acids Res 2008 Jan 36 1 e5 2 Alarge genome center s improvements to the Illumina sequencing system Quail et a Nat Methods 2008 Dec 5 12 1005 10 3 Amplification free Illumina sequencing library preparation facilitates improved mapping and assembly of G C biased genomes Kozarewa et al Nat Methods 2009 Apr 6 4 291 5 4 Titration free massively parallel pyrosequencing using trace amounts of starting material Zheng et al Nucleic Acids Res 2010 Apr 30 5 Rapid quantification of DNA libraries for next generation sequencing Buehler et al Methods 2010 Apr 50 4 S15 8 6 A scalable fully automated process for construction of sequence ready barcoded libraries for 454 Lennon et al Genome Biol 2010 11 2 R15 Version 1 10 15
3. number of libraries that you are able to quantify per kit depends on how many libraries you will be processing at one time and on the qPCR format that you will be using i e single tubes 48 96 or 384 well plates By way of analogy the situation is similar to that of a simple DNA marker ladder for agarose gels if you run many samples per gel then the DNA ladder will be sufficient for many samples but if you run only one sample per gel then the DNA ladder will be finished quickly A single KAPA Library Quantification Kit is sufficient for the quantification of 30 libraries following our recommended protocols in a 96 well format More libraries can be quantified Using a 384 well plate format smaller reaction volumes and or fewer replicates Many variables will greatly affect the ultimate number of libraries that can be quantified per kit Below are a few guidelines based on the details of our recommended protocols iii Only one preferably triplicate set of six DNA standards needs to be run per assay f you quantify a single library sample at a time you will be able to quantify six libraries per Kit of DNA standards Each quantification experiment will use up 6 x 3 qPCR reactions for the standards plus 4 x 3 qPCR reactions for the library sample 30 qPCR reactions in total Since each Kit contains 5 mL KAPA SYBR FAST 2X qPCR Master Mix sufficient for 500 x 20 pL reactions you will have 500 uL 6x 30 uL 320 qPC
4. qPCR Master Mix and the Primer PreMix would go twice as far the volume of DNA Standard added to each reaction remains 4 uL regardless of the reaction volume v We recommend that you perform serial dilutions of each library sample This means that each library sample gives rise to 4 different dilutions There are two reasons for this First it helps to ensure that at least one dilution will fall within the upper limit of the range of the assay Second it allows calculation of the qPCR efficiency for that particular library sample which is not required for quantification but helps with trouble shooting Thus it is possible that with experience and or with very standardized workflows you may feel confident to stop doing these serial dilutions in which case the number of qPCR reactions required for each quantification will be reduced vi While we recommend triplicate reactions for the DNA standards and for the Library samples some users may feel sufficiently confident especially after some experience with the kit to do away with some replicates of the standards and or the samples Of course this would make it difficult or impossible to troubleshoot any unexpected results and the quantification accuracy may suffer On the other hand many more libraries could be quantified with the same amount of reagent 4 What are the primer sequences used in the various KAPA Library Quantification Kits Roche 454 FLX Primer Premix KAPA product KP00
5. relatively inexact science qPCR performs better than any of the alternatives e g Bioanalyzer spectrophotometry for limiting variations in cluster density or bead enrichment Of course if you are working with very variable sample types source organism fragment sizes etc and library prep techniques then you may have to do more cluster amplification titrations or accept more variability in cluster density or bead enrichment We have found that qPCR generally yields somewhat lower concentrations for a given library sample than would be obtained using less specific quantification methods such as spectrophotometry or electrophoresis As a point of reference one customer performing highly automated human re sequencing has found that DNA input at 10 pM determined using the KAPA Library Quantification Kit for the Illumina sequencing platform consistently yields cluster densities around 220 000 clusters tile In the case of 454 emPCR it seems that optimal input concentrations range between 0 1 1 copies per bead when libraries are quantified using qPCR as opposed to inputs between 1 and 10 copies per bead when spectrophotometry and or electrophoresis are used for library quantification Version 1 10 4 S KAPABIOS Inconsistencies in emulsion formation may contribute to the broad range of potentially optimal input concentrations required for the 454 FLX and Titanium platforms 3 How many libraries can I quantify with a single kit The
6. to use correspondingly less DNA according to the qPCR determined concentration in order to generate the equivalent cluster density or bead enrichment percentage Of course we would expect qPCR quantification to result in less variability of cluster density or bead enrichment because it minimizes at least one potential source of that variability the percentage of nucleic acid in the sample that is not amplifiable by PCR However there are other sources of variability for which qPCR cannot account e g pipetting inaccuracies Furthermore it is worth considering that both cluster amplification and bead emPCR are expected to be less efficient than standard solution phase PCR as in qPCR and one might therefore expect cluster amplification and bead emPCR to be more sensitive to difficult templates For this reason even though qPCR is the most appropriate surrogate for cluster amplification titrations or emPCR titrations the relationship between measured library concentration and cluster density or bead enrichment may Version 1 10 2 S KAPABIOS be influenced by such factors as GC content secondary structure template length DNA damage etc You may therefore need to use different amounts of DNA to achieve equivalent cluster density or bead enrichment depending on the organism being sequenced the sample type the library construction method etc However with time and experience it should be possible to gain a feel for how the
7. 01 FLX Primer A 5 GCC TCC CTC GCG CCA 3 FLX Primer B 5 GCC TTG CCA GCC CGC 3 Roche 454 Titanium Primer Premix KAPA product KP0002 Titanium Primer A 5 CCA TCT CAT CCC TGC GTG TC 3 Titanium Primer B 5 CCT ATC CCC TGT GTG CCT TG 3 Illumina GA II IIx Primer Premix KAPA product KP0003 Primer P1 5 AAT GAT ACG GCG ACC ACC GAG 3 Primer P2 5 CAA GCA GAA GAC GGC ATA CGA 3 Illumina GA II IIx Primer Premix KAPA product KP0005 Primer P1a 5 AAT GAT ACG GCG ACC ACC GA 3 Primer P2 5 CAA GCA GAA GAC GGC ATA CGA 3 Life Technologies SOLID Primer Premix KAPA product KP0004 Lib PCR Primer 1 5 CCA CTA CGC CTC CGC TTT CCT CTC TAT G 3 Lib PCR Primer 2 5 CTG CCC CGG GTT CCT CAT TCT 3 Version 1 10 6 X KAPABIOS 5 Whatis the primary reason for poor reproducibility across replicate data points Inaccurate pipetting is the most common reason for poor reproducibility Accurate quantification requires careful pipetting For liquid handling systems consult the relevant user manual Alternatively apply the following when using a non automated device Examine the tip before dispensing to ensure that the correct volume is being added Flush rinse the tip 2 3 times after dispensing Use a new pipette tip every time After thawing and mixing briefly centrifuge reagents to prevent droplets on tube walls from transferring to the outside of the pipette tip Try to
8. A molecules uL Std 6 0 0002 pM 20 81 120 dsDNA molecules uL Life Technologies ABI SOLiD DNA Standard Std 1 10 0000 pg uL 3 74 63 424 344 dsDNA molecules uL Std 2 1 0000 pg pL 7 08 6 342 434 dsDNA molecules uL Std 3 0 1000 pg uL 10 41 634 243 dsDNA molecules uL Std 4 0 0100 pg uL 13 74 63 424 dsDNA molecules uL Std 5 0 0010 pg uL 17 07 6 342 dsDNA molecules uL Std 6 0 0001 pg uL 20 41 634 dsDNA molecules uL Tube code Length Molecular weight dsDNA 454 FLX DNA Standard KQO001 486 bp 299 66 kDa 454 Titanium DNA Standard KQO0002 459 bp 282 98 kDa Illumina GA Standard KQ0003 452 bp 277 43 kDa SOLID DNA Standard KQ0004 154 bp 94 95 kDa Version 1 10 S KAPABIOSYSTEM 8 Why does the recommended cycling protocol in the KAPA Library Quantification Kit protocol differ from that in the KAPA SYBR FAST qPCR Kit protocol Next generation sequencing libraries are generally complex comprising a very wide diversity of DNA fragments While KAPA SYBR FAST qPCR reagents are generally capable of extremely fast thermocycling we conservatively recommend relatively long denaturation and annealing extension times for library quantification in order to accommodate the diversity of templates in a typical library sample The recommended qPCR protocol for library quantification consists of an initial denaturation step at 95 C for 5 min followed by 35 cycles of denaturation at 95 C for 30 sec and combined annealing extension at 60 C for 45 sec
9. I original 454 FLX libraries Yes No 454 FLX Titanium Rapid Prep libraries No Yes 454 FLX Titanium Lib L libraries All standard Titanium libraries made by ligation of adaptors to library No Yes fragments Amplicon libraries for unidirectional sequencing 454 FLX Titanium Lib A libraries Amplicon libraries for bidirectional Yes No sequencing As noted in the table above our Library Quantification Kit for the Titanium platform is compatible with the Titanium Rapid Prep libraries but we suggest that customers use a longer combined annealing extension time in the cycling protocol to accommodate the longer fragment sizes that are often used with these libraries see FAQ 1 above Version 1 10 13 S KAPABIOS If you know the adaptor or Fusion Primer sequences used to make your library then it is best to check them against the following qPCR primer sequences to ensure compatibility with one of our two 454 Library Quantification Kits KAPA Library Quantification Kit for 454 FLX platform FLX Primer A 5 GCC TCC CTC GCG CCA 3 FLX Primer B 5 GCC TTG CCA GCC CGC 3 KAPA Library Quantification Kit for 454 Titanium platform Titanium Primer A 5 CCA TCT CAT CCC TGC GTG TC 3 Titanium Primer B 5 CCT ATC CCC TGT GTG CCT TG 3 3 What are the advantages and disadvantages of using the fluorescently labeled adaptor supplied in the Roche FLX Titanium Rapid Library Preparation Kit for library q
10. Note The Ct values in the table below are arbitrary and will vary significantly based on the qPCR instrument and threshold setting used Version 1 10 KAPA e Typical Ct on Given Conc Rotorgene dsDNA mol uL 0 1 threshold Roche 454 FLX DNA Standard Std 1 100 000 000 ssDNA molecules uL 2 06 50 000 000 dsDNA molecules uL Std 2 10 000 000 ssDNA molecules uL 5 39 5 000 000 dsDNA molecules uL Std 3 1 000 000 ssDNA molecules uL 8 72 500 000 dsDNA molecules uL Std 4 100 000 ssDNA molecules uL 12 06 50 000 dsDNA molecules uL Std 5 10 000 ssDNA molecules uL 15 39 5 000 dsDNA molecules uL Std 6 1 000 ssDNA molecules uL 18 72 500 dsDNA molecules uL Roche 454 Titanium DNA Standard Std 1 100 000 000 ssDNA molecules uL 2 13 50 000 000 dsDNA molecules uL Std 2 10 000 000 ssDNA molecules uL 5 46 5 000 000 dsDNA molecules uL Std 3 1 000 000 ssDNA molecules uL 8 79 500 000 dsDNA molecules uL Std 4 100 000 ssDNA molecules uL 12 13 50 000 dsDNA molecules uL Std 5 10 000 ssDNA molecules uL 15 46 5 000 dsDNA molecules uL Std 6 1 000 ssDNA molecules uL 18 79 500 dsDNA molecules uL Illumina GA DNA Standard Std 1 20 0000 pM 4 14 12 044 283 dsDNA molecules uL Std 2 2 0000 pM 7 48 1 204 428 dsDNA molecules uL Std 3 0 2000 pM 10 81 120 443 dsDNA molecules uL Std 4 0 0200 pM 14 14 12 044 dsDNA molecules uL Std 5 0 0020 pM 17 47 1 204 dsDN
11. R reactions left over when you have finished the DNA Standards In this case additional DNA Standards can be purchased separately so that the leftover qPCR reagent is not wasted f you have many libraries to quantify simultaneously and run 96 well qPCR plates then you can load the 30 reactions described above 18 Standards and 12 samples plus additional library samples on the same qPCR plate Since there are 66 wells available you can load 66 12 5 additional library samples on the plate for a total of 6 libraries plate In this case you will quantify 6 x 6 36 libraries before the DNA Standards are used up Each plate will use 18 triplicate standards plus 6 x 12 72 library samples which is 90 qPCR reactions Therefore you will require 90 x 6 540 qPCR reactions in total which means that you could expect to run out of qPCR Master Mix more or less at the same time as you run out of DNA Standards As mentioned above if you run full 96 well plates exactly according to the recommended protocol with 6 libraries per plate then a single KAPA Library Quantification Kit should allow the quantification of 30 libraries Similarly it is possible to use 384 well plates or 48 well plates and in each case the cost library sample will be different Version 1 10 5 e KA PA iv While the recommended qPCR volume is 20 uL many users may choose to run 10 uL volumes especially in 384 well formats in which case the KAPA SYBR FAST
12. S IKAPABIOS KAPA Library Quantification Kits Frequently Asked Questions Standard protocols for all three major commercial Next Generation Sequencing NGS platforms employ unreliable laborious and costly methods for quantifying library DNA molecules prior to clonal amplification of sequencing templates to form polonies polymerase colonies Accurate quantification of bona fide PCR competent sequencing templates is crucial for reliable clonal amplification via bridge PCR DPCR cluster amplification or emulsion PCR emPCR underestimation results in non clonality and or over clustering while overestimation leads to poor yields of clusters or template carrying beads Most standard methods for quantifying NGS libraries have a number of important disadvantages First electrophoresis and spectrophotometry measure total nucleic acid concentrations whereas optimal cluster density or template to bead ratio depends on the appropriate input concentration of PCR amplifiable DNA templates Since the proportion of amplifiable DNA molecules in a library may vary with each sample expensive and time consuming titrations are required Second these methods have low sensitivity consuming nanograms of precious samples or about 1000 times more molecules than are required for sequencing Finally electrophoresis and spectrophotometry are not suited to high throughput of samples requiring laborious and error prone manual liquid handling In princ
13. Standards used in our Library Quantification Kits are not sequencing libraries which are impossible to manufacture reproducibly through multiple production lots and over extended periods of time Instead we use a defined pure linear dsDNA amplicon for each set of DNA Standards This allows us to rigorously validate their efficiency and reproducibility for use as qPCR amplification standards Before accepting a newly manufactured lot into our inventory we use a stringent qPCR assay to compare each new lot of KAPA Library Quantification DNA standards to a reference set of standards during manufacturing and quality control We compare Ct scores for each standard in a newly manufactured set with Ct scores in a reference set of standards and we ensure that each standard lies within 0 1 Ct of the respective reference standard and that the resulting standard curve essentially lies on top of the reference standard curve minimal deviations in y intercepts and slopes Version 1 10 11 S IKAPABIOS FAQs specific to Roche 454 FLX Titanium sequencing platforms 1 Are the KAPA Library Quantification Kits for the Roche 454 Titanium platform compatible with the Rapid Library Preparation Kit Yes our Library Quantification Kit for the 454 Titanium platform is compatible with the Rapid Prep libraries Although the adaptor moeity used in the Rapid Prep kits is different to the adaptors used in the standard Titanium library prep they share the same cor
14. ant to consider implementing a larger up front dilution of your library samples in your standard work flow If the first standard is omitted ensure that the library Ct score falls within the dynamic range of Standards 2 6 7 What are typical Ct values for the standards When examining the data from the DNA standards one should look at two things first i Are the replicates very close to one another A difference of 1 Ct is equivalent to a 2 fold difference in template concentration In our hands using a Corbett Rotorgene 6000 HRM qPCR instrument we would expect to see less than 0 1 Ct difference between replicates More variance than this may indicate a large degree of well to well variation in the instrument optics and or temperature control or problems with pipetting accuracy Is there consistent even spacing between consecutive DNA standards and is the spacing close to the expected 3 32 cycles The DNA standards should amplify with close to 100 efficiency and therefore consecutive standards 10 fold dilutions should be separated by 3 32 cycles in Ct scores ACt If ACt is consistently different from 3 33 this may indicate problems either with pipetting accuracy or problems with the efficiency of the qPCR reaction While it is acceptable to discard the occasional outlier a large number of outliers generally indicates a problem either with the qPCR instrument or with the liquid handling equipment and or technique
15. avoid placing the pipette tip too far under the surface when aspirating as this may result in additional liquid adhering to the outside of the tip Dispense directly into the bottom of the tube or well Ensure that no residual liquid remains in the tip after dispensing Another common cause of poor consistency among replicates is incompletely thawed DNA template standards or library samples please ensure that all reagents are completely thawed and thoroughly mixed before assembling your qPCRs Instrument variability may also contribute to poor consistency across replicates please ensure the qPCR instrument has been properly calibrated 6 l am concerned that the Ct of DNA Standard 1 and or some of my library samples is too low amplification is too early In developing the KAPA Library Quantification Kit we aimed to provide an assay with the broadest possible dynamic range However depending on the specific qPCR instrument and user defined settings for the instrument it is possible that amplification of the most concentrated DNA standard Std 1 may give rise to an increasing fluorescent signal during the automatic baseline determination performed by the qPCR instrument Many instruments use the early cycles to calculate and set the baseline subtraction correction and samples that are changing significantly in fluorescence during this phase can dramatically affect this process since there is no stable baseline This also applie
16. both ends of the size distribution In summary some initial work will be required to determine optimal loading concentrations for each type of library sample when customers switch from Bioanalyzer and or dye assisted spectrophotometry Because qPCR quantifies a specific subset of the total DNA molecules present and because of relatively large variability in Bioanalyzer quantification accuracy direct comparisons between the two methods may not be particularly useful Version 1 10 3 S IKAPABIOS Based solely on the inherent variability in quantification methods it is probably reasonable to expect up to 2 fold difference in either direction between quantification by qPCR or by Bioanalyzer or similar Moreover for the reasons discussed above it is reasonable to expect qPCR would generally register significantly lower concentration than Bioanalyzer for a given library sample Assuming no technical problems the qPCR method for quantifying sequencing libraries should provide the most reliable predictions of cluster density or bead enrichment 2 What is the recommended concentration of input DNA for optimal cluster density or template to bead ratio We expect that the KAPA Library Quantification Kits should allow users to dispense with cluster amplification titrations or emPCR titrations almost entirely once they are comfortable with qPCR quantification of their libraries Indeed many high throughput users cannot possibly perform clusterin
17. e sequences used for amplifying the library fragments onto the beads during emPCR and for subsequent sequencing Our qPCR primers bind to these core emPCR sequences in the library fragments However please note that the longer fragment lengths enabled by the Rapid Prep kits may require some optimization beyond our standard protocols When using qPCR to quantify libraries of larger fragment sizes in the new Rapid Prep libraries you may experience suboptimal qPCR amplification efficiencies The engineered polymerase used in KAPA SYBR FAST reagents displays greater affinity for DNA and correspondingly greater processivity so it should outperform standard qPCR reagents in this regard Nevertheless as the target length increases amplification may become progressively more vulnerable to inhibition by other factors such as high or low GC content secondary structure and DNA damage When using qPCR to quantify libraries with fragment sizes above 700 bp we suggest that you increase the annealing extension step in the recommended thermocycling profile as follows Initial denaturation 95C 5 min Cycling x35 cycles Denaturation 95C 30sec Annealing Extension 60C 90sec We also recommend that you pay particular attention to quantifying 2 fold dilution series of these samples and then use the resulting qPCR data to calculate the reaction efficiency If you find that the large fragment libraries are not amplifying efficiently compared to the s
18. eristics of your melt curves might correlate with problems such as adaptor dimers Please note that performing melt curve analysis on the Illumina DNA Standards will reveal a multiple peak The multiple peak is the result of differential local melting in the linear amplicon due to a heterozygous SNP and is not indicative of non specific amplification 10 What are the storage and stability recommendations for KAPA Library Quantification Kits and the combined KAPA SYBR FAST Primer Mix KAPA Library Quantification Kits are shipped on dry ice or ice packs depending on the country of destination Upon receipt store the entire kit at 20 C in a constant temperature freezer When stored under these conditions and handled correctly all kit components will retain full activity for at least six months from the date of receipt All components of the KAPA Library Quantification Kits as well as the combined KAPA SYBR FAST Primer Premix solution are stable through more than 30 freeze thaw cycles We therefore recommend that all reagents are stored in the dark at 20 C when not in use Nevertheless these reagents are stable in the dark at 4 C for at least one week and may be stored in this state for short term use provided that they are not contaminated with microbes and or nucleases 11 How are the DNA standards in the KAPA Library Quantification Kits quality controlled to minimize batch to batch variance and ensure reliability The DNA
19. es related to instrument settings data collection or data processing analysis Apart from any initial larger dilutions that may be required we recommend that the sample be diluted in a 2 fold dilution series Assuming the same pipette is used for both sample and diluent an accurate 2 fold dilution should be produced even if pipetting is systematically inaccurate e g the pipette is not accurately calibrated Again we recommend that dilutions be performed in triplicate and the resulting data should allow outliers to be identified and discarded and should help to identify any problems arising from pipetting accuracy or well to well variability iii Bioanalyzer accuracy the stated coefficient of variation for quantification using the relevant Bioanalyzer DNA assays is 20 Moreover in our experience Bioanalyzer assays are prone to erratic behavior depending on instrument maintenance reagent age and storage conditions operator error etc Other factors that may affect quantification by Bioanalyzer relate to the size distribution of library fragments it is possible that the size cut offs used to define the peak to be quantified might exclude fragments especially at the smaller end which would give rise to clusters or to enriched beads Similarly a relatively small change in the position of the calculated baseline and or the detection limit of the instrument assay may under or over estimate a significant number of library fragments at
20. fication with the aim of predicting cluster density or bead enrichment Aside from errors in data collection and processing i e qPCR instrument and software settings issues or data analysis i e calculations performed by the user there are three obvious possible explanations for such differences i qPCR counts only those library molecules that are competent templates for PCR and is therefore blind to all library molecules that cannot give rise to clusters during the bridge PCR process of cluster amplification Illumina GA or to beads carrying amplified sequencing template during emPCR Roche 454 and Life Technologies SOLiD We have therefore found that qPCR usually provides a lower estimate of library concentration than the less specific methods such as Agilent Bioanalyzer or dye assisted spectrophotometry e g Picogreen DNA molecules in the sample may not support PCR for a number of reasons a they may not carry the appropriate adaptors at both ends b they may contain difficult sequences high AT or GC content and or structures which inhibit or prevent PCR c they may be damaged in some way that prevents PCR amplification e g nicks or UV induced cross linking note exposure to UV especially in the presence of ethidium bromide will damage DNA in ways that will prevent PCR amplification If qPCR is consistently providing lower estimates of library concentration than your previous previous method s then you would need
21. g emPCR titrations on all of their library samples and they rely solely on qPCR to determine optimal DNA input for cluster amplification or emPCR Nevertheless some facilities especially those that process a wide variety of libraries representing a range of sample types library construction procedures etc may find that titrations are still sometimes necessary and or useful In such cases qPCR is nevertheless likely to be the most informative quantification method prior to carrying out the titrations and for inferring optimal loading concentrations for subsequent similar samples Inter laboratory differences across various sample types library construction methods handling etc make it difficult to provide specific recommendations about the relationship between optimal library concentration and cluster density bridge PCR or template to bead ratio emPCR In the case of the Illumina sequencing platforms the ideal cluster density varies from user to user and has been trending upwards with upgrades to hardware and software Furthermore the relationship between input DNA concentration and cluster density bead enrichment is not necessarily linear If your lab is following a standard sample preparation workflow then we would expect that you should quickly be able to use your own experience to determine the best average DNA concentration that yields the required cluster density or template to bead ratio in your hands While this remains a
22. ification for use in sequencing This presents additional pipetting steps and opportunities for sample confusion contamination and loss ii The standard curve generated in the Rapid Library Prep method covers 8 x 2 3 dilutions from 2 5 x 10 mol uL down to 1 46 x 10 mol uL The KAPA Library Quantification Kits cover a dynamic range across six orders of magnitude from 5 x 10 mol uL down to 500 mol L Version 1 10 14 S IKAPABIOS iii For quantification using the Roche Rapid Prep method the user is required to produce the standard curve making 7 serial 2 3 dilutions of the supplied standard Aside from the extra work this entails this also presents additional scope for variability and errors The KAPA Library Quantification Kit contains pre prepared and validated standards for generating the entire standard curve FAQs specific to Illumina GA sequencing platform 1 Compatibility of KAPA Library Quantification Kit with various types of libraries on Illumina platform KAPA Library Quantification Kits for the Illumina GA sequencing platform kit codes KK4824 KK4835 KK4844 KK4854 KK4808 KK4809 containing Primer Premix tube code KP0005 are compatible with all Illumina GA library types Note Previous versions of the kit KK4822 KK4832 KK4842 KK4852 KK4804 KK4805 containing Primer Premix tube code KP0003 were not compatible with small RNA or GEX Digital Gene Expression Taq Profiling library adaptor sequences We will
23. iple quantitative PCR qPCR is inherently well suited for NGS library quantification and overcomes many of the difficulties presented by the standard approaches qPCR specifically quantifies PCR competent DNA molecules is accurate across a very broad dynamic range is amenable to automated liquid handling and is cost effective Moreover because qPCR is extremely sensitive it allows accurate quantification of very dilute libraries and consumes small amounts of sample Because qPCR allows reliable cluster amplification or bead emPCR from dilute samples less PCR amplification of libraries is required and the biases and loss of complexity associated with library amplification can be minimized There are two important considerations for reliable qPCR quantification of library DNA i Ideally qPCR amplification should be efficient across the broad range of templates that constitute sequencing libraries Traditional qPCR reagents are optimized for efficient amplification of short ideal amplification targets In contrast factors such as target length unbalanced GC content and problematic secondary structures may lead to low amplification efficiency resulting in unreliable quantification of an indeterminate proportion of library molecules ii lt is important to use reliable calibration standards that display minimal variability from lot to lot and over long periods of time To address these specific requirements of NGS library qua
24. ntification KAPA Library Quantification Kits comprise highly consistent sets of serially diluted DNA standards and state of the art qPCR reagents which include a DNA polymerase specifically engineered for robust SYBR Green I tolerant amplification of long and difficult templates Version 1 10 1 e KA PA 1 Why is there a difference between the concentrations obtained using the qPCR based KAPA Library Quantification Kit method and the Agilent Bioanalyzer Invitrogen Qubit spectrophotometry based assay Many users of the qPCR based KAPA Library Quantification Kits LQK notice significant differences between the concentrations determined for a given sample using qPCR versus other methods such as the Agilent Bioanalyzer For many of the reasons stated below we believe that qPCR quantification of libraries is the best approach for minimizing variability in cluster density or bead enrichment In this respect consider that the actual values obtained for the concentrations of libraries is ultimately irrelevant for the reliable generation of desirable cluster densities or bead enrichments rather a reliable relative quantification of libraries with respect to one another and in relation to their potential for producing clusters or templated beads is critical While Bioanalyzer assays may remain a useful tool for assessing library quality mainly with respect to size distribution we do not believe that they provide the best method for quanti
25. omplexity a few dominant library fragments rather than a huge number of unique fragments Although NGS library samples are expected to cross the cycle threshold Ct relatively early the typical cycling profile for qPCR Library Quantification may run for 30 or 35 cycles The melt curve analysis will only be performed after the cycling is completed so one must keep in mind that the sample being analyzed during melting has undergone a large number of cycles Many artifacts may be generated during these periods of extensive amplification and subsequent thermocycling and these artifacts may be evident in the melt curve analysis and or after electrophoresis gel or Bioanalyzer even though they may not be representative of the quality of the starting material used in the qPCR Despite the aforementioned considerations we have received feedback from researchers who claim to detect adaptor dimers in their SOLID libraries by melt curve analysis after qPCR We would therefore recommend that gel electrophoresis or a Bioanalyzer assay be used to check on the quality of your libraries and follow the melt curve analysis too If your Version 1 10 10 S IKAPABIOS library samples are consistently crossing the Ct relatively early then you might choose to reduce the cycle numbers of your qPCR accordingly in order to reduce the possibility of artifacts as described above With some experience and trial and error you may be able to determine which charact
26. s to the first DNA standard if the raw data amplification plot increases visibly within the first 5 6 cycles then this may be an issue Unfortunately it is not possible for us to give precise guidelines on this as it is an instrument specific issue and in many cases depends on user specific instrument settings Please consult your qPCR instrument manual for more information It is usually possible to tell whether this is an issue by examining the amplification plots if the corrected processed amplification plot begins well below the baseline of samples crossing the Ct later then it is likely that baseline determination subtraction was not successful for that plot Another useful way to judge whether an early Ct value has created problems during data analysis is to confirm that the expected spacing between consecutive standards is 3 32 cycles the standards represent a 10 fold dilution series Similarly consecutive two fold dilutions of a given sample should cross the Ct 1 cycle apart Version 1 10 7 KAPABIOSYSTEMS If you feel that the first standard and or some sample dilutions have Ct scores that are too early then simply exclude that standard and those sample dilutions from the analysis This is most easily done by de selecting them in the qPCR analysis software If you experience this issue regularly with your instrument settings workflow then you may choose to omit the first DNA standard from all future assays and you might w
27. se factors influence the relationship between library concentration and cluster density or bead enrichment ii Pipetting accuracy is a common source of error in all library quantification methods especially when small volumes are measured and when serial dilutions are performed Because qPCR is highly sensitive requires only tiny amounts of sample and utilizes low reaction volumes all advantages in other respects it is particularly important to pay close attention to the quality and condition of the equipment used as well as to the techniques employed We have attempted to minimize the possible effects of inaccurate pipetting in the following ways Strictly validated DNA Standards are supplied in a pre prepared 10 fold dilution series and we recommend that 4 uL of each sample be used per reaction to avoid pipetting of small volumes Pipetting accuracy will affect the accuracy of the standard curve only in the setting up of qPCR reactions 4 uL template and 16 pL Master Mix We recommend that new users set up reactions with the DNA Standards in triplicate as this will help to identify outliers and to indicate whether pipetting accuracy at this point in the workflow is a potential problem It should also be possible to use these data to distinguish between systematic pipetting inaccuracy vs random errors in pipetting accuracy The triplicate standard curve data may also help to identify well to well variability in the qPCR instrument and issu
28. tandards then you may try the following approaches i Increase the combined annealing extension time by a further 30 seconds and or ii Compensate for the reduced efficiency of sample amplification via calculation Version 1 10 12 e KA PA 2 The KAPA Library Quantification Kit for the Roche FLX platform is recommended for Roche 454 GS Titanium amplicon libraries For historical reasons KAPA Biosystems supplies two sets of 454 Library Quantification Kits one set is for the original FLX adaptor sequences and the other set is for the newer Titanium adaptor sequences Late in 2009 Roche 454 released the Rapid Library Preparation Kit employing a single adaptor moiety in place of the traditional adaptor sequences Around the same time the company released Titanium amplicon sequencing reagents using FLX derived Fusion Primer sequences and then subsequently released single read Titanium amplicon sequencing protocols that use Titanium derived Fusion Primers These developments have created significant confusion about which KAPA Library Quantification kit should be used with each of the various possible Titanium libraries Please consult the following table to ensure that you obtain the appropriate KAPA Library Quantification Kit Compatible KAPA Library Quantification Kit Type of Roche 454 library for 454 FLX platform for 454 Titanium platform KK4820 KK4830 KK4821 KK4831 KK4841 KK4840 KK4850 KK4851 AI
29. uantification in comparison with qPCR Roche 454 has released a library preparation protocol with new reagents which is called the GS FLX Titanium Rapid Library Preparation Kit This kit makes the preparation of 454 sequencing libraries quicker and easier The new kit uses a single adaptor moiety in place of the two distinct adaptors used before Roche has addressed library quantification by incorporating a fluorescent label into the new adaptor in the Rapid Library Prep Kits This fluorescent label increases the sensitivity of detection so that less material is used up during library quantification and it should help to address the problem of incorrectly counting library molecules that do not carry appropriate adaptors for emPCR a major issue for Bioanalyzer and intercalating dye assisted spectrophotometry However it is still possible that an unknown and variable proportion of library molecules will carry an adaptor on only one end and may not be amplifiable during emPCR for other reasons GC content secondary structures etc In addition to the advantage of qPCR outlined above i e it counts only bona fide amplifiable templates users may find value in the following i Although improved quantification by the standard Rapid Library Prep method is still not very sensitive and requires large volumes 20 50 pL of undiluted library sample This sample must therefore be recovered from the cuvette or multi well plate after quant
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