Designing Your ChIP-seq Experiments: The Data is in the Details

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One question that many people have when transitioning to NGS for their chromatin immunoprecipitation (ChIP) experiments is what parameters need to be considered in the experimental design phase in order to produce optimal data quality. The good news is that ChIP-seq has become a widely used assay and multiple resources are available to assist in nearly every step of the process. Here we will discuss several frequently asked questions and important details that should be considered in the experimental design, sequencing, and analysis of ChIP-seq projects. Continue reading…

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16S rRNA Amplicon Sequencing Offers Enhanced Metagenomic Detection

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Using 16S ribosomal RNA (rRNA) gene sequencing to identify and characterize microbial populations has greatly increased our understanding of the role microbes play in environmental, agricultural and health-related settings. The 16S rRNA gene is comprised of highly conserved regions flanking nine hyper-variable regions, which are ubiquitous in bacterial species. The following data illustrates how the NEXTflex™ 16S V1-V3 Amplicon-Seq Kit, combined with the Illumina MiSeq, provides users with a time-efficient and robust method to study bacterial metagenomics, using any sample from which DNA can be isolated.

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Optimized Library Prep for 16S V1 – V3 rRNA Sequencing for Improved Bacterial Metagenomics Studies

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Bioo Scientific recently launched the NEXTflex™ 16S V1 – V3 Amplicon-Seq Kit for bacterial metagenomics analysis. This kit facilitates the preparation of multiplexed amplicon libraries that span the V1-V3 hypervariable regions of bacterial 16S ribosomal RNA (rRNA) genes, producing multiplexed libraries compatible with paired-end sequencing on the MiSeq Illumina® sequencing platform. The NEXTflex 16S V1 – V3 Amplicon-Seq Kit is ideal for bacterial diversity studies, studies of interactions between host species and bacterial communities, identification of non-culturable bacteria, detection of adventitious agents and enzyme discovery and production. With the ability to multiplex up to 384 samples, the NEXTflex 16S V1 – V3 Amplicon-Seq Kit is optimized to simplify and improve your bacterial metagenomics analysis.

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The Role of Circulating Tumor DNA Analysis in Early Detection and Treatment of Cancer

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The existence of extracellular cell free DNA (cfDNA) was detected in blood as early as 1948. In 1977 it was documented for the first time that levels of cell free tumor DNA (cftDNA) were significantly higher in cancer patients than in healthy persons. In the last decade, human whole genome sequencing has revealed the relationship between somatic mutations in oncogenes, tumor suppression genes, and cancer development. This led to the rediscovery of cftDNA as a valuable resource for diagnosis, prognosis, treatment decisions, and follow-up monitoring of cancer patients. Next generation sequencing has led to a number of interesting findings that could pave the way for doctors to use less invasive techniques for cancer detection, offering actionable diagnostic information at an early stage of cancer development.

In February of 2014, a group from Johns Hopkins presented a comprehensive report in Science Translational Medicine which moved the application of ctDNA analysis as a cancer diagnostic to a new level (1). This study analyzed ctDNA from 640 patients with 18 cancer types at four clinical stages, and was able to present several interesting conclusions:

 

  • ctDNA could be detected in more than 50% of localized tumors and more than 75% of advanced tumors. In most tumor types, levels of ctDNA correlated with the stage of cancer.
  • Detection of cancer based on ctDNA analysis is more sensitive than analysis of circulating tumor cells (CTC). ctDNA and associated mutations were always detected at higher levels in blood of cancer patients, and could even be detected when CTC were absent. This observation suggests that ctDNA does not originate from CTC.
  • ctDNA and localized tumors had the same mutations and/or rearrangements, confirming that most ctDNA is shed to the bloodstream by tumor cells.
  • It is possible to predict the resistance to EGFR blockage treatment of colon cancer by acquisition of new mutations in KRAS, BRAF, NRAS and EGFR, which could help tailor personal treatment during disease progression.
  • 47% of patients with stage I disease had detectable ctDNA in plasma.

 

The most promising finding from this research was that mutations found in ctDNA could be used to detect and treat colon cancer during stage I growth. However, many problems have to be solved before a ctDNA test can be used for routine clinical diagnosis. Currently, it is difficult to determine the origin and location of a tumor from analysis of ctDNA in the blood of an asymptomatic person, because the majority of known mutations occur in many different types of tumors. Moreover, as few as 50 million malignant cells can produce detectable amounts of cftDNA in blood (2), which is far below the detection limit of current imaging methods. Hopefully, the results presented will encourage more prospective studies using analysis of cftDNA by next generation sequencing to produce highly informative data, which will help narrow the suspected origin of mutated ctDNA and allow intervention prior to the progression of disease.

In conclusion, this report validates ctDNA analysis for tumor diagnosis as a promising, non-invasive method for screening, diagnosis, treatment decisions and monitoring of human cancer. The spread of cftDNA analysis promises to generate further information for broad implementation into clinical applications.

Bioo Scientific developed the NEXTflex™ Cell Free DNA-Seq Kit to facilitate the analysis of cfDNA for studies such as these. The NEXTflex Cell Free DNA-Seq Kit has been optimized for the construction of DNA-seq libraries from cell-free fetal or circulating tumor DNA. With a short, two hour DNA library prep protocol, this 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 workflow and shortens hands-on library construction time. In addition, the availability of up to 192 unique adapter barcodes facilitates high-throughput applications.

 

(1)    Bettegowda C. et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med 2014; 6: 224a24.

(2)    Diaz LA Jr. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancer. Nature 2012; 486:537-540.

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Preventing Cross Contamination of Samples and Adapter Barcodes during NGS Library Prep

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Because NGS libraries are usually multiplexed, with multiple libraries run in a single well, preventing cross contamination during library preparation and sequencing is a critical issue. The following Tech Tips, Preventing Cross Contamination of Samples and Adapter Barcodes during NGS Library Preparation, identifies areas of possible contamination and suggests techniques to use to prevent cross contamination. – Josh Kinman

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Achieving High Coverage and Yield from GC and AT Rich Genomes

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The PCR amplification step in library preparation introduces biases including uneven coverage of regions with extreme base composition, increased numbers of duplicate fragments, decreased mapping quality and poor variant calling. GC-rich regions are particularly intractable, being difficult to uniformly PCR amplify. Unbiased amplification of highly complex whole genome sequencing (WGS) libraries, whole exome sequencing (WES) libraries and targeted re-sequencing libraries is necessary in order to obtain high quality data for de novo genome assembly and accurate calling of single nucleotide polymorphisms (SNPs). In our new white paper, we show that the NEXTflex™ PCR polymerase contains a highly optimized and robust enzyme that exhibits minimal GC bias and produces uniform coverage of difficult to sequence genomes.

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Improve Your RRBS or WGBS Library Diversity

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The NEXTflex™ Bisulfite-Seq Kit is a versatile kit designed for both reduced representation bisulfite sequencing (RRBS) and whole-genome bisulfite sequencing (WGBS), yielding highly diverse libraries with uniform coverage for Illumina® sequencing. The NEXTflex™ U+ Polymerase Mix included in the kit is able to read through uracil present in DNA templates and has been optimized to amplify bisulfite converted templates. This kit also features Enhanced Adapter Ligation Technology, resulting in library preps with greater library diversity and a larger number of unique sequencing reads. This specially designed NEXTflex ligation enzymatic mix allows users to perform ligations with longer adapters and superior ligation efficiencies.

The availability of the methylated NEXTflex™ Bisulfite-Seq barcoded adapters makes multiplexing simple.

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Check Out Our New Look

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Bioo Scientific is introducing updated NGS kit boxes! These new boxes for NGS are smaller in size for easier storage in crowded lab shelves, and the improved utilization of space in the new boxes allows us to ship many of our kits in one box instead of two. The redesign features recyclable inserts and a scannable QR code leading to our new feedback form. We look forward to hearing from you about your experience with our kits.

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Streamlined Library Construction for Quantitative, Directional, and Standard RNA-Seq

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RNA Sequencing (RNA-Seq) is a valuable tool for a broad range of clinical, environmental, and basic research.  Producing high quality RNA-Seq libraries can be challenging for several reasons, including isolation of pure RNA, efficiently converting RNA to cDNA, and loss of material incurred during the series of enzymatic steps and cleanups required for library construction. Here we introduce Bioo Scientific’s family of Illumina compatible NEXTflex™ Rapid RNA-Seq kits, all of which include the thermostable NEXTflex™ Rapid Reverse Transcriptase enzyme. The NEXTflex Rapid RNA-Seq Kits provide affordable and unique technology, some of the shortest RNA-Seq library preparation times of any kits on the market, include all enzymes required for library preparation, and produce the highest quality RNA-Seq library. Read the entire white paper here.

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Tech Tips – Barcode Recommendations for Low Level Multiplexing

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To reduce costs of NGS, libraries are often prepared using molecular barcodes, also known as indices. The NEXTflex™ Barcodes contain index sequences, short sequences of 6 – 12 bases that are incorporated into each library to allow several libraries to be multiplexed and sequenced together on a single flow-cell of a sequencer. When barcoded NGS libraries are sequenced, each cluster generated on an Illumina sequencer includes its own unique barcode sequence, which is then associated with a specific library. Barcodes can be introduced into the library during the adapter ligation step (by including them as a subset of the adapter sequences), or they can be introduced during the subsequent PCR step, by including them as a subset of the Forward or Reverse PCR primers. For DNA and mRNA library prep Bioo Scientific recommends introducing the barcodes during the adapter ligation step of library construction. This avoids problems due to amplification bias caused by PCR primer sequences, and allows use of multiplexing when making libraries that do not include a PCR step.

Low-plex multiplexing (i.e. mixing up to ~ 12 samples) is often chosen as a reasonable compromise between low cost/low per sample coverage, and high cost/high coverage. High levels of multiplexing can involve use of 96 or more different barcodes, usually using commercially available barcode sets such as Bioo Scientific’s NEXTflex-96™ Barcodes. With high level multiplexing, sufficient diversity in the large library of barcode sequences is ensured. In contrast, when using low-level multiplexing, it is possible that the barcode sequences chosen could lack sufficient diversity to avoid “registration failure” on an Illumina sequencer. Registration failure could occur if the color balance was not maintained between the red and green lasers (used to sequence A/C bases and G/T bases, respectively). Barcode sequences for low-level multiplexing need to be chosen such that each position of the barcode will result in signal in both the red and green color channels. In other words, each position in the set of index sequences needs to include at least one A or C base, at least one G or T base, and ideally an equal balance of both.

 

Examples of acceptable combinations of barcodes for 2-way multiplexing:

GCCAAT and CTTGTA   and   ACAGTG and GGTCAA

(every position in each pair has at least one A/C and one G/T)

Examples of unacceptable barcode combinations for 2-way multiplexing:

GCCAAT and ACAGTT   and   ACAGTG and CTTGTA

(first pair lacks G/T at positions 2 and 3 and lacks A/C at position 6; second pair lacks G/T at first position, lacks C/A at fourth and fifth positions)

 

With these considerations in mind, Bioo Scientific recommends the use of specific barcode combinations for low-level multiplexing. These low-level multiplexing recommendations can be found in Appendix A in all of our barcode manuals. Note that recommendations are given for low-plex barcode combinations even for our larger barcode sets, to provide flexibility for use in situations where low-plex multiplexing is desirable. - Dr. Marianna Goldrick

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