- generation sequencing (FA-NGS) library preparation. - Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process.. - We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. - Conclusions: We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. - Therefore, we speculate that the FA-NGS workflow has less risk of user error. - While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.. - 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Department of Energy Agile BioFoundry, Emeryville, CA, USA Full list of author information is available at the end of the article. - Each of the aforementioned quantification methods have been validated for NGS library preparation, but are notably la- borious when applying them to high-throughput work- flows. - We call this library preparation workflow modi- fication fluorescent amplification for NGS (FA-NGS).. - In addition to reducing the number of steps in the NGS library preparation workflow, qPCR with SYBR green I added supplementary benefits to the modified NGS work- flow. - We first tested FA-NGS with Illumina’s Nextera XT (Illumina, San Diego, CA). - We next evaluated FA-NGS with adapter ligation-based library construction kit using the NEBNext Ultra II DNA library preparation kit. - The libraries from both modified workflows utilizing FA- NGS (Fig. - 1, Additional file 1: Fig. - We chose to apply FA-NGS to two commonly used methods for preparing libraries. - Using an input titration assay, we deter- mined that the concentration of input adapter-ligated (AL) gDNA or plasmid could be tracked by FA-NGS with NGS primers (Fig. - 2, Additional file 2: Fig. - S2 and Additional file 3: Fig. - We observed that we could apply Nextera tagmentation and FA-NGS to plasmids with starting amounts as low as 1.5 pg per 7.5 μL reac- tion. - We also found that the detection limit of FA-NGS above a 1000 end relative fluorescence unit (RFU). - For gDNA samples, the end RFU values scaled with the 2-fold dilution factor of the start- ing amount of the input (Fig. - MCA of the AL gDNA input showed that even samples with in- put below 62.5 pg still have amplified DNA, as observed by a high melting temperature peak (Fig. - 2c, Additional file 4: Fig. - End RFU values were used as a proxy for relative concentration of each individual library. - 3b, Additional file 4: Fig. - MCA (Additional file 5: Fig. - S5 and Additional file 6:. - 2c, Additional file 3: Fig. - 1 FA-NGS Workflow: Following library preparation method of choice, amplification is applied with SYBR green mastermix, including polymerase (pink clouds), intercalating dye (green rectangles), and index primers (yellow, blue, purple rectangles). - Melting curve analysis is then applied to an aliquot of the library to determine amplification success. - 2 Input titration of AL-DNA monitored with FA-NGS: DNA diluted 2-fold starting at 1000 pg per reaction was amplified with SYBR green in duplicate and monitored with continuous fluorescence (a), end-fluorescence (b), and melting curve analysis (c). - To determine whether the end RFU values were a reason- able measurement of the final concentration of DNA after amplification, we sequenced the two libraries consisting of 96 combinations of dual indices with the Illumina MiSeq Reagent Nano kit. - Both the Nextera and AL libraries yielded similar distribution of percent reads with a p -value of 1 (Additional file 7: Fig. - While each sample is not pooled at exactly 1.04% of the reads (expectation for optimal pool- ing from 96 samples), a majority of the samples from each library do fall below a 50% difference from optimal pooling range (Additional file 8: Fig. - Sequencing quality value scores for the PhiX Control Library and for the FA-NGS li- braries were above the specification provided by Illumina of at least 80% Q30 [22] (Additional file 9: Fig. - Table 1 Plasmids used in Nextera library preparation. - All plasmids used are available through the public instance of the ABF registry [18]. - 4 Library pooling yields near even distribution of percent of the total reads: Optimal pooling is the expectation (1.04) of the percent reads if all 96 libraries pooled evenly. - The bars are colored by the end RFU values of the qPCR, blue if the RFU was greater than 1000, yellow if the RFU was between 700 and 1000, and red if the RFU was less than 700. - For gDNA library (b), samples below RFU 700 were likely to be underpooled because they were out of the linear range of the qPCR. - FA-NGS allows for accurate pooling directly after PCR, reducing the risk of user error. - Typical NGS library prep- aration requires individual library purification prior to QC and pooling (Additional file 1: Fig. - FA-NGS resolves these problems with a sin- gle step of amplification and quantification.. - Many workflows, es- pecially Nextera-based library preparations lack QC tests until the final step of the protocol. - When used on small aliquots of the amplified libraries, MCA confirms if an amplified library product is present. - In order to make this modified workflow accessible to researchers with high- throughput NGS workflows, we made a FA-NGS soft- ware tool available on GitHub (see availability of data and materials) that can be used for set-up, analysis, and pooling of FA-NGS libraries. - of the end-fluorescent values and small multiple MCA plots in the plate layout of the user’s choice. - Because of the potential variation in DNA shearing or fragmentation size distributions from NGS library preparation, it may be recommended to both measure the library concentrations and perform individual library size analysis to enable pool- ing in equimolar amounts. - 4, Additional file 7:. - S7 and Additional file 8: Fig. - S8) from using the FA- NGS library quantification strategy is sufficient to give comparable representation of 96 libraries.. - To further validate the FA-NGS workflow, we performed sequencing quality assessment and included the Illumina PhiX Library Control for comparison. - All libraries surpassed the recommended quality value score (Additional file 9: Fig.. - S9), per Illumina’s specification of the percentage of bases at or above Q30, indicating that the FA-NGS workflow suc- cessfully generated high quality sequencing reads.. - While implementing FA-NGS has many benefits, the ap- parent pitfalls must be noted. - Because these samples were out of the fluorescence linear range of the qPCR, it is likely that the end-fluorescent values for these samples could not be used as a precise proxy for the relative DNA concentration and were therefore transferred at a volume that overestimated the relative concentration. - We speculate that variation in percent reads for plasmid libraries could be an effect of amplifying beyond the exponential phase to the plateau phase of qPCR, such that the end-fluorescent values used for pooling were less precise (Additional file 10:. - For standard library preparation workflows that use conventional PCR, there could be a comparable limita- tion during the final library quantification step, depending on the fluorescence linear range of the DNA quantification assay [25, 26]. - give relatively low read counts compared to other Illu- mina sequencing kits, they generated more than suffi- cient coverage for the 96 plasmids (Additional file 11:. - We demon- strate the FA-NGS workflow ease of use with fewer overall steps than conventional library workflows, as well as an MCA QC test to confirm successful library construction before sequencing. - An open source FA-NGS software tool is available to assist in implementing the workflow (see availability of data and materials). - We expect that the ap- plication of FA-NGS will greatly benefit the production of any NGS library type which is amplified by PCR.. - Nextera library amplification of using real-time qPCR Pairwise 8-nucleotide barcodes and the remainder of the Illumina adapter sequences were added using primers from the Nextera XT Index Kit v2. - Real-time qPCR and concur- rent amplification and relative quantification of the library was facilitated on the CFX384 Touch Real-Time PCR De- tection System using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA). - Reaction volumes were 7.5 μL total, with 0.25 μL each of the indexed Nex- tera primers, 3.75 μL of SYBR Green, 2.25 μL water, and 1 μL of the previous reaction. - The end RFU values were determined with CFX Manager software (Bio- Rad, Hercules, CA) for all libraries.. - Size dis- tribution of the adapter ligated DNA was verified on the Bioanalyzer (Agilent, Santa Clara, CA). - The end RFU values were determined with CFX Manager software (Bio-Rad, Hercules, CA) for all libraries.. - 7 μL of the diluted PCRs were transferred to low dead volume (LDV) plates (Labcyte, Sunnyvale, CA) compatible with the Labcyte Echo 550 (Labcyte, Sunnyvale, CA) leaving 3 μL of diluted PCR behind. - The transfer volume of each PCR reaction was calcu- lated with the end RFU values determined with CFX Manager Software (Bio-Rad, Hercules, CA). - Each well was transferred to a 384-well PCR plate (Bio-Rad, Hercules, CA) with the Echo 550 (Labcyte, Sunnyvale, CA) using the transfer volumes calculated from the end RFU values.. - FA-NGS software tool. - The FA-NGS software tool was written in python using pandas, numpy, seaborn, and matplotlib.pyplot libraries.. - The FA-NGS software tool was written in python and is available for download. - Additional file 1: Figure S1. - Standard NGS and FA-NGS Workflow Comparison: Side-by-side comparison of the Standard NGS workflow (left) and modified FA-NGS workflow (right) highlights how FA-NGS can save time and hands on steps in preparing NGS libraries. - Additional file 2: Figure S2. - Input titration of Nextera library prepared plasmid monitored with FA: DNA diluted 4-fold starting at 100 pg per Nextera tagmentation reaction was amplified with SYBR green and moni- tored by continuous fluorescence (A), and melting curve analysis (B) Additional file 3: Figure S3. - MCA maxima correlates with input titration: Amount of input DNA is correlated with the local maxima of the MCA determined derivative RFU of the input titrations of both Nextera (A) and AL (B) library prepared samples, with R2 equal to 0.807 and 0.842 respectively. - Additional file 4: Figure S4. - Distributions of end RFU and transfer volumes of AL libraries: End RFU values (A) and transfer volumes (B) of AL libraries generated with 500 (blue), 250 (red), 125 (yellow), and 62.5. - Additional file 5: Figure S5. - Additional file 6: Figure S6. - Additional file 7: Figure S7. - The range of percent reads for the AL library (red) was with a mean of 1.04 and a standard deviation of 0.5 Additional file 8: Figure S8. - Additional file 9: Figure S9. - Sequencing quality scores of Nextera and AL libraries: The percentage of bases with ≥ Q30 quality score for PhiX Control Library and for Nextera and AL libraries demonstrates sequencing quality for FA-NGS libraries. - Additional file 10: Figure S10. - Additional file 11: Table S1. - Additional file 12: Table S2. - FA-NGS: Fluorescent amplification for next generation sequencing;. - We would like to thank Cindi Hoover and Samuel Deutsch from the DOE Joint Genome Institute, who performed early development of the Nextera workflow. - JC and MG both performed library preparation and sequencing. - This work was part of the Agile BioFoundry (http://agilebiofoundry.org) supported by the U.S. - This work was also part of the ENIGMA, Ecosys- tems and Networks Integrated with Genes and Molecular Assemblies (http://. - The views and opinions of the authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. - The FA-NGS software tool was written in python and is available for down- load at: https://github.com/AgileBioFoundry/FA-NGS. - All plasmids used are available through the public instance of the ABF registry: (https://public- registry.agilebiofoundry.org/folders/2) [18]. - Miniaturization and optimization of 384-well compatible RNA sequencing library preparation.
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