NEXTflex™ Cell Free DNA-Seq Library Prep Kit for Illumina® Sequencing

Ideal for sequencing of cfDNA and ctDNA on an Illumina® sequencer

Catalog# Product Name Quantity US List Price
5150-01 NEXTflex™ Cell Free DNA-Seq Kit
8 rxns $303 Buy Now
5150-02 NEXTflex™ Cell Free DNA-Seq Kit
48 rxns $1,362 Buy Now
514101 NEXTflex™ DNA Barcodes - 6 48 rxns $466 Buy Now
514102 NEXTflex™ DNA Barcodes - 12
96 rxns $919 Buy Now
514103 NEXTflex™ DNA Barcodes - 24
192 rxns $1,821 Buy Now
514104 NEXTflex™ DNA Barcodes - 48
384 rxns $3,582 Buy Now
514105
NEXTflex-96™ DNA Barcodes - 96 768 rxns
$6,926 Buy Now
514160
NEXTflex™ Dual-Indexed DNA Barcodes 768 rxns $6,926 Buy Now
514161
NEXTflex™ Dual-Indexed DNA Barcodes 768 rxns $6,926 Buy Now

NEXTflex cf/ctDNA Sequencing Kit for Illumina

  • Designed for low sample input - Only 1 ng of input DNA required
  • Accelerated workflow requiring as little as 2 hours, with minimal hands-on time
  • Enhanced Adapter Ligation Technology offers a larger number of unique sequencing reads
  • Flexible adapter barcode options – up to 384 unique barcodes are available
  • Compatible with Illumina® sequencing platforms
  • Protocols available for automation

 

Bioo Scientific’s NEXTflex™ Cell Free DNA-Seq Kit can produce libraries from 1 ng of cell free DNA for circulating tumor DNA (ctDNA) or cell free fetal DNA (cffDNA) analysis, in two hours. This low-input library preparation kit delivers high coverage quality and reduced bias for Illumina sequencing applications.

This kit is for research use only. Not for use in diagnostic procedures.


Reduced Bias and Coverage Gaps

NEXTflex Cell Free DNA-Seq Kits contain NEXTflex™ PCR polymerase, a high fidelity enzyme that exhibits minimal GC bias and produces uniform coverage of difficult to sequence genome regions. This kit also incorporates Bioo Scientific’s patent pending Enhanced Adapter Ligation Technology, which offers the best available ligation efficiency, resulting in greater library diversity and a larger number of unique sequencing reads. These optimizations enable researchers to obtain more information from each sequencing run.


Prepare Libraries in Only 2 Hours

The NEXTflex Cell Free DNA-Seq Kit can be used to prepare single, paired-end and multiplexed DNA libraries for sequencing using Illumina® platforms. The NEXTflex™ 1-step End-Repair and Adenylation protocol simplifies the library prep workflow and shortens hands-on library construction time. In addition, the availability of up to 384 single-index barcodes facilitates high-throughput applications.

We recommend using the NextPrep-Mag™ cfDNA Isolation Kit for isolation of circulating DNA from blood plasma prior to library prep with the NEXTflex Cell Free DNA-Seq Kit.


Automation Protocols Available

A protocol is currently being validated for the PerkinElmer Sciclone Workstations. Contact us at NextGen@biooscientific.com for more details.


Flexible Multiplexing Options


The NEXTflex Cell Free Illumina DNA-Seq Library Prep Kit is designed to be used with the NEXTflex™ DNA BarcodesNEXTflex-96™ DNA barcodes and NEXTflex-HT™ Barcodes. These barcodes are available in sets of 6, 12, 24, 48, 96 and 384 unique indexed adapters. All of these NEXTflex™ Adapters are long, annealed adapters that offer an improved multiplexing workflow and flexible setup.


Papers that Cite the Use of the NEXTflex Cell-Free DNA-Seq Kit:

Xia, S. et al. (2015) Genomic variations in plasma cell free DNA differentiate early stage lung cancers from normal controls. Lung Cancer. doi: 10.1016/j.lungcan.2015.07.002.


Kit Specs

The NEXTflex Cell Free DNA-Seq Kit for Illumina Library Prep contains enough material to prepare 8 or 48 DNA samples for Illumina® sequencing. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20°C. These kits ship on dry ice. 

NEXTflex Cell Free DNA-Seq Kit Protocol for Illumina® Sequencing

 

NEXTflex Cell-Free DNA-Seq Workflow for Illumina

MAKING CELL-FREE DNA-SEQ LIBRARIES 

To assess performance of the NEXTflex™ Cell Free DNA-Seq Kit, plasma was obtained from a commercial source (HemaCare® Corp) and from healthy consented volunteers. DNA was extracted using a magnetic bead-based protocol from 0.6 mL plasma samples. The cfDNA was eluted from the magnetic beads in 50 µL volume, and 3 – 4 µL was used to verify cfDNA recovery. Then 32 µL of the recovered cfDNA was used as input to make barcoded DNA-Seq libraries using the NEXTflex Cell Free DNA-Seq Kit. Libraries were amplified using various numbers of PCR cycles, and analyzed on agarose gels and Agilent® BioAnalyzer® high-sensitivity DNA chips. Since cell-free plasma DNA is naturally fragmented, no further fragmentation was needed prior to library construction. In some cases, the cell-free DNA was enriched for small fragments (reflecting the smaller size of fetal DNA) before library construction. Control libraries were produced using 1 ng of sheared genomic DNA and analyzed in conjunction with the cell-free DNA libraries. Select libraries were sequenced on an Illumina® NextSeq® instrument, as 2 x 150 paired-end reads. The entire process of extracting cfDNA and creating libraries from multiple plasma samples can be easily completed in a single day.

RESULTS

A. Assessment of Libraries Prior to Sequencing

12 µL (~35%) of each library were run on the gel. Outer lanes are 100 bp ladder run as molecular weight marker. Libraries were made using 15 cycles of PCR. Lanes 1 – 4 show libraries from Donors 1 – 4. Donor 2 was from a woman in third trimester of pregnancy. Sample in lane 5 was made from DNA recovered from re-elution of the magnetic beads from Donor 2, in 100 µL additional Elution Solution (optional Step 11 in kit protocol). Lane 6 shows a library made from 1 ng of sheared human genomic DNA. Note the distinct size distribution of libraries made from cell-free DNA, which reflects its origin from apoptotic cells.

 

Assessment of  cell free DNA library products

Figure 1. Assessment of library products by ethidium bromide staining.


Assessment of cell free DNA  libraries on Agilent BioAnalyzer

Figure 2. Assessment of libraries on Agilent BioAnalyzer High Sensitivity DNA Chip


Panel A: Library from 32 µL (64% of prep) of cell-free DNA from a male donor amplified for 15 cycles. Note the broad size distribution, which reflects the discrete sizes of cell-free DNA fragments. The cell-free DNA was not fragmented prior to use.
Panel B: Analysis of the corresponding input cell-free DNA used to make the library shown in Panel A. Note, the concentration of cell-free DNA is too low to be detected, which is typical.
Panel C: Library made from 1 ng of sheared human genomic DNA, amplified for 15 cycles. Note the much different size distribution compared to the library made from cell-free DNA.
Panel D: Library made from cell-free DNA size-selected prior to library construction, using Ampure magnetic beads to enrich for small cell-free DNA. Library was amplified for 12 cycles. Sample was from a male donor. After adapter ligation, the desired library products are approximately 300 bp.

B. NGS Results

Two cell-free plasma DNA libraries, one derived from a healthy male donor and one from a healthy third-trimester pregnant woman carrying a male fetus, were submitted for paired end 150 base shotgun sequencing to the core sequencing facility at University of Texas, Austin. Both libraries were from total cell-free DNA (not size selected) and were amplified for either 12 cycles (Library 14) or 13 cycles (Library 22). The libraries were analyzed at the sequencing facility to verify that they were of adequate concentration and size distribution prior to sequencing, and both libraries passed these quality metrics. The QC results for Library 22 are shown below. Each library was successfully sequenced in two separate multiplexed sequencing runs, on the Illumina HiSeq and on the Illumina NextSeq.


QC results for cell free DNA libr

Figure 3. Pre-sequencing QC results for sample Library 22

Number of peaks found: 14   Noise: 0.3   Corr. Area 1: 600.0
Region table for sample 1    From [bp] To [bp] 234 - 4,442  Corr. Area: 600.0  % of Total: 94
Average Size [bp]: 615     Size distribution in CV [%]: 72    Conc. [pg/μl]: 519.50  Molarity [pmol/l]: 1,811.7 


Several key metrics of library quality, shown in Table 1, are the percentage of reads that are due to adapter contamination, the % of reads that are attributable to PCR duplicates, and the % of reads that map to the human genome and to each chromosome. The libraries had low levels (1.1% and 4.9%) of uninformative reads representing adapter dimers (unwanted side products consisting of 5’ adapter ligated to 3’ adapter). The proportion of reads mapping to the human genome was quite high (close to 100%). The percentage of reads attributed to PCR duplicates (defined as reads with identical sequences at the 5’ adapter and 3’ adapter junctions) was higher in Library 22 (58%) than in Library 14 (16%), possibly reflecting the greater number of PCR cycles used to produce Library 22. Even so, after filtering PCR duplicates, Library 22 still had > 12 million reads, which was sufficient for analysis, as over 97% were mapped to the genome.  

The metrics shown in the graphic below for Library 14 depict extremely high-quality sequencing data, as shown by the high Phred scores (Y-axis). The trend line shows average scores above 30, corresponding to >99.9% probability of accuracy, for the average of all reads in the position ranges corresponding to cfDNA sequence (as depicted along the X-axis), with the exception of the longest reads (positions 145-150), which have slightly lower scores (but which are still above 99% probability of being accurate). Results for Library 22 were similar.

Q uality scores for cell free DNA libraries


% Duplication = (Reads after trimming adapter - Reads after filtering duplicates)/Reads after trimming adapter
% Mapping = (Reads mapping to genome/Reads after filtering duplicates)

Figure 4. Quality scores across all bases. (Sanger/Illumina 1.9 encoding)


% of adapter contamination and reads mapping to human genome

Table 1. Low % of adapter contamination and high % of reads mapping to human genome

The results of mapping the reads from each library to each human chromosome are shown in Table 3. There is generally very good agreement between the observed proportion of the total reads that map to each chromosome and the expected proportion based on the size of each chromosome. Of particular interest are the reads mapping to the Y chromosome in Library 14, which was made from cfDNA from a woman carrying a late gestational age (3rd trimester) male fetus; reads in Library 14 that map to Y chromosome are derived from fetal DNA. The number of reads mapped to the Y chromosome in Library 14 (319,835 reads) represent 0.66% of the total number of reads (48,387,865). Since the total size of the human genome is 3,095,677,412 bp and the total size of the Y chromosome is 59,373,566 bp, the Y chromosome represents 1.92 % of the human genome. This value is in good agreement with the observed % of reads mapping to the Y chromosome (1.79 %) in Library 14, which was made from cfDNA from a male donor. Extrapolating from these values, we calculate that 34% of the cfDNA in Library 14 is derived from fetal DNA.

Cell free DNA library reads mapping to each chromosome

Table 2. Reads mapping to each chromosome 


DISCUSSION/CONCLUSIONS

Results presented herein demonstrate the performance of the NEXTflex Cell Free DNA-Seq Kit for processing cell-free DNA for NGS applications. Recovery of a high percentage of fetal DNA in the maternal plasma sample was shown by the analysis of the proportion of reads mapping to the Y chromosome in the library derived from late-gestational stage plasma; these reads represent DNA from the male fetus that is present in the maternal circulation. The value we report for % fetal DNA (34%) is on the upper end of values reported in other studies. Some of these reports were based on analyses using different techniques, but one study that used NGS reported a similar high level of fetal DNA (40%) (Fan et al 2010). The authors speculated that the relatively high % of fetal DNA in their studies was due to the higher efficiency of amplification of the shorter fetal cfDNA fragments, compared to the longer maternal cfDNA fragments, during the library amplification step. For non-invasive prenatal diagnostics, magnetic bead-based size selection is sometimes used prior to library construction to enrich for the shorter fragments found in maternal plasma. In this case, the size distribution of the predominant library product reflects that expected for fetal DNA. The distribution of the library products we observed using unfractionated cfDNA shows several peaks, which probably reflects the fact that most cfDNA in healthy donors is thought to originate from the “ladder” of DNA fragments derived from apoptotic cells. It is interesting that a significant fraction of the library products in Library 22, from a healthy male donor, are quite large, ranging up to several kilobases. The proportion of larger library products may be increased in plasma from cancer patients.

The NEXTflex Cell Free DNA-Seq Kit is optimized for library construction from low input amounts of ctDNA or cfDNA isolated from cell free fluids. The NEXTflex Cell Free DNA-Seq Kit can be used to construct Illumina-compatible libraries from 1 ng of DNA in about two hours. This kit delivers high coverage and reduced bias, along with flexible multiplexing options. The high quality of the DNA-Seq libraries produced using this kit is demonstrated by several metrics of sequencing quality including a high % of reads mapping to the human reference genome, low levels of adapter contamination, Phred scores indicative of extremely accurate base-calling, and excellent correspondence between observed and expected % of reads mapping to each human chromosome.

References

1. Chan KCA, Zhang J, Hui ABY, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92.
2. Dennis Lo YM, Chiu RW. Prenatal diagnosis: progress through plasma nucleic acids. Nat Rev Genet 8:71–7 (2007).
3. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Nat Acad Sci U S A;105:16266–71(2008).
4. Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W,Leung TY, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Nat Acad Sci U S A 105: 20458–63 (2008).
5. Chiu RW, Sun H, Akolekar R, Clouser C, Lee C, McKernan K, et al. Maternal plasma DNA analysis with massively parallel sequencing by ligation for noninvasive prenatal diagnosis of trisomy 21. Clin Chem 56:459–63 (2010).
6. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, and Quake SR. Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing Clinical Chemistry 56:8 (2010)
7. Wang BG, Huang H-Y, Chen Y-C, Bristow RE, Kassauei K, Cheng C-C, Roden R, Sokoll LJ, Chan DW, and Shih l-M.  Increased Plasma DNA Integrity in Cancer Patients. CANCER RESEARCH 63, 3966–3968, July 15, 2003
8. Gautschi, O. et al. Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Cancer Lett. 254, 265–273 (2007).
9. Kuang, Y. et al. Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistant non-small cell lung cancer. Clin. Cancer Res. 15, 2630–2636 (2009).
10. Leary, R.J. et al. Development of personalized tumor biomarkers using massively parallel sequencing. Sci. Transl. Med. 2, 20ra14 (2010).
11. McBride, D.J. et al. Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors. Genes Chromosom. Cancer 49, 1062–1069 (2010).
12. Taniguchi, K. et al. Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas. Clin. Cancer Res. 17, 7808–7815 (2011).
13. Forshew, T. et al. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci. Transl. Med. 4, 136ra168 (2012).
14. Leary, R.J. et al. Detection of chromosomal alterations in the circulation of cancer patients with whole-genome sequencing. Sci. Transl. Med. 4, 162ra154 (2012).
15. Dawson, S.J. et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N. Engl. J. Med. 368, 1199–1209 (2013).
16. Diehl F, and Smergeliene E. BEAMing for Cancer: Detecting Tumor Mutations in Peripheral Blood Using Digital PCR. Genetic Engineering and Biotechnology News Vol 33 No 15 (2013).
17. Murtaza, M. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013).
18. Crowley, E., Di Nicolantonio, F., Loupakis, F. & Bardelli, A. Liquid biopsy: monitoring cancer-genetics in the blood. Nat. Rev. Clin. Oncol. 10, 472–484 (2013).
19. Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA,  Merritt RE, Shrager JB, Loo BW Jr, Alizadeh AA, and Diehn M. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nature Medicine, Received 15 August 2013; accepted 6 November 2013; published online 6 April 2014; doi:10.1038/nm.3519
20. Diaz LA and Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. Journal of Clinical Oncology/American Society of Clinical Oncology 32:6 Feb 20 2014.

Kit Contents

NEXTflex™ End-Repair & Adenylation Buffer Mix

NEXTflex™ End-Repair & Adenylation Enzyme Mix

NEXTflex™ Ligase Enzyme Mix

NEXTflex™ DNA-Seq Adapter 1

NEXTflex™ PCR Master Mix

NEXTflex™ Primer Mix

Nuclease-free Water

NEXTflex™ Resuspension Buffer

 

Required Materials Not Provided

 ~1 ng of Cell-Free DNA in up to 32 µL nuclease-free water.

NEXTflex™ DNA Barcodes – 6 / 12 / 24 / 48 (Cat # 514101, 514102, 514103, 514104) or NEXTflex-96™ DNA Barcodes (Cat # 514106)

Ethanol 100% (room temperature)

Ethanol 80% (room temperature)

96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar

96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) or similar

Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)

Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63880)

Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar

Thermocycler

2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes

Nuclease-free barrier pipette tips

Vortex