Nextera library preparation technology used to characterize HIV intra-host diversity

A new publication from the David O’Connor laboratory at UW-Madison describes the use of Nextera™ technology to sequence whole HIV and SIV genomes.

The authors sequenced virus from an Indian rhesus macaque experimentally infected with SIVmac239 and coding regions from 11 HIV-positive patients. Overlapping RT-PCR amplicons were used to cover the virus genome sequences, and the RT-PCR-amplified genomes were simultaneously fragmented and tagged using the Nextera Roche 454-Compatible Enzyme Mix, followed by pyrosequencing. An average of 41,826 sequence reads per SIV genome was obtained, with an average coverage depth of 380 sequences. For HIV samples, an average of 29,000 sequence reads per genome with a sequencing depth range of 208-846 was obtained. Full or near-full coverage was obtained with Nextera libraries prepared from 50 ng of DNA.

The authors conclude that, using Nextera technology, they are able to demonstrate:

“...a new and highly practical approach to study the complexity of the viral population within a host and identify minor variants on a genome-wide scale. While this manuscript applies pyrosequencing to immunodeficiency viruses, this approach could be applied to any viral pathogen.”

Bimber, B. et al. (2010). Whole genome characterization of HIV/SIV intra-host diversity by ultra-deep pyrosequencing Journal of Virology DOI: 10.1128/JVI.01378-10

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Nextera libraries from buccal-cell DNA

We’ve received several inquiries regarding the use of Nextera™ technology to prepare next-generation sequencing libraries from buccal-cell DNA. The BuccalAmp™ DNA Extraction Kit is designed for a quick extraction of DNA for PCR only. However, our R&D scientists have developed a protocol, outlined below, to ensure successful preparation of Nextera libraries from DNA extracted with the BuccalAmp Kit. For this example, DNA was extracted from four buccal swabs and pooled.

1. Centrifuge the tube briefly (1 minute at 3,000 x g) to remove the solid cellular debris.
2. Remove the supernatant and precipitate the DNA with 1/10 volume of sodium acetate (3.0 M) and 2 volumes of ethanol.
3. Resuspend the pellet in 100 µl of T₁₀E₁ buffer. Centrifuge briefly to remove insoluble material and transfer the supernatant to a DNA Clean & Concentrator-5 column (Zymo).
4. Add 300 µl of Binding Buffer and follow the manufacturer’s protocol.
5. Elute the DNA with two aliquots (10 µl each) of T₁₀E₁ buffer. Use 50 ng of eluted DNA, as determined spectrophotometrically, in the standard Nextera protocol.

Human buccal DNA after tagmentation reactions with the Nextera™ DNA Sample Prep Kit (Illumina-Compatible).  DNA was extracted and cleaned up as described above, and treated as follows: lane 1, no treatment ; lane 2, Hind III digestion; lane 3, Nextera tagmentation with HMW buffer; lane 4, Nextera tagmentation with LMW buffer. Lane M, 100-bp DNA ladder.

Size distribution of Nextera Illumina-compatible libraries

Many of our Nextera™ customers have requested the ability to prepare libraries containing different sizes of DNA fragments for Illumina GAII sequencing. The Nextera™ DNA Sample Prep Kit (Illumina-compatible) now contains two buffers, Low-Molecular-Weight Buffer (LMW) and High-Molecular-Weight Buffer (HMW). The size distribution of the tagmented DNA can be controlled based on the buffer used in the reaction. Below are two Bioanalyzer traces from standard Nextera reactions using the indicated buffer.

LMW Buffer

HMW Buffer

We have also further optimized the reaction conditions to yield a narrow size distribution. For additional information on reaction conditions and on bias, library complexity, coverage, etc., please contact our technical support staff: techhelp@epibio.com

HPV prevalence in esophageal squamous cell carcinoma

Esophageal cancer is currently the eighth most common human cancer, with esophageal squamous cell carcinoma (ESCC) being the most common subtype. Tobacco and alcohol use are the most prevalent causes of ESCC; however, limited evidence suggests that infectious agents--in particular, human papillomavirus (HPV)--are linked to ESCC. Antonsson et al. recently analyzed HPV prevalence and lifestyle factors in ESCC patients. Archived tumor samples from a nationwide cohort of 222 ESCC patients in Australia were tested for the presence of HPV DNA by PCR, and positive samples were sequenced to determine HPV type. DNA was extracted from FFPE tissue blocks or slides using the QuickExtract™ FFPE DNA Extraction Kit. Samples were analyzed for the presence of HPV with general mucosal HPV primers, and β-globin PCR primers were used as a control. Of the 222 ESCC patients, only eight tested positive for HPV (six cases of HPV-16; two cases of HPV-35). None of 55 esophageal tissue controls from healthy patients tested positive for HPV. Lifestyle factors were also investigated in this study. Overall, there was weak evidence that that patients with HPV-positive ESCC had higher BMI than patients with HPV-negative tumors. The authors conclude that larger studies or pooled analyses will be required for definitive evidence regarding the role of HPV in ESCC.

Antonsson, A. et al. (2010). High-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma Cancer Epidemiol Biomarkers Prev, 19 (8), 2080-2087 DOI: 10.1158/1055-9965.EPI-10-0033