Understanding the 454 Method

A DNA strand is prepared by cutting the sample into small fragmented pieces (Fig 1). Attached to the ends of these fragments are oligonucleotide adaptors (Fig 2). These allow the fragments to individually attach to primer-coated beads. The goal is to have one fragment per bead. Amplification essentially copies fragments on each bead (Fig 3). Beads are then filtered, ridding of unattached DNA fragments. For sequencing, a single bead is placed into a picotiter-volume well on a plate accompanied with an enzyme bead to be incubated. Nucleotide bases are released in waves. A light signal is generated when each base is incorporated. The intensity of light is proportional to the number of repeated nucleotides of the same type.

Many DNA and genome sequencing laboratories can provide services like this. MR DNA Lab is a next generation sequencing service provider and bioinformatics service provider specializing from 454 pyrosequencing to the new Ion Proton sequencing platform. They have over 20 years of continuous experience (rather than combined experience) developing new and novel molecular methods. Sytematics, microsatellite screening, MHC assays, viral assays, protozoan assays and etc. Visit www.mrdnalab.com for more information, or find them on Facebook and Twitter.

Cancer genomics at MR DNA

Cancer is a type of disease in which cells divide abnormally without control and are able to invade other tissues. One person dies from cancer each minute in the United States. As the population ages, this number is expected to increase. Thus, we need to understand cancer to control and ultimately conquer it. There are more than 100 different types of cancer. Cancer is a genetic disease that can be caused by many changes across the genome. Cancer cells can spread to other parts of the body through the blood and lymph system. As cancer progresses, cells accumulate additional somatic mutations and propagate to form new cancer clones. As a result, most advanced cancers are polyclonal. Monitoring transcriptome and epigenome changes in cancer cells can help answer questions about disease classification, prognosis, and progression (Mardis and Wilson, 2009; Boehm and Hahn, 2011) and there are a variety of sequencing approaches that enable researchers to detect these changes. Next generation sequencing (NGS) has been instrumental in advancing scientific fields related to human disease. Advances in NGS technology are enabling the systematic analyses of whole cancer genomes, providing insights into the landscape of somatic mutations and the great genetic heterogeneity that defines the unique signature of an individual tumor (Wong et al., 2011). We use Illumina’s TruSeq Amplicon – Cancer Panel (TSACP) for cancer genomics studies, a highly multiplexed targeted resequencing assay for detecting somatic mutations. TSCAP provides predesigned, optimized oligonucleotide probes for sequencing mutational hotspots in >35 kilobases (kb) of target genomic sequence. Within a highly multiplexed reaction, 48 genes are targeted with 212 amplicons. The assays begin with hybridization of the pre-mixed, optimized oligonucleotide probes upstream and downstream of the regions of interest. Each probe includes a target capture sequence and an adapter sequence used in a subsequent amplification reaction. An extension- ligation reaction extends across the region of interest, followed by ligation to unite the two probes. Extension-ligation templates are PCR amplified and two unique sample-specific indices are incorporated. The final reaction product contains amplicons that are ready for sequencing (figure 1). TSACP enables highly sensitive mutation detection within important cancer-related genes, including BRAFKRAS, and EGFR. Mutations in these genes are linked to many cancers, including melanoma, colorectal, ovarian, and lung cancer. The analysis of next-generation sequencing data from cancer samples can be challenging. MR DNA offers a number of software options and analysis tools to simplify this process. We at MR DNA routinely perform cancer panel targeting resequecning assay for detecting somatic mutations with large volume of samples and provide cost effective high quality data and robust output from only low input DNA.

References:

  1. Wong KM, Hudson TJ, McPherson JD. Unraveling the genetics of cancer: genome sequencing and beyond. Annu Rev Genomics Hum Genet. 2011;12:407-430.
  2. Boehm JS, Hahn WC. Towards systematic functional characaterization of cancer genomnes. Nat Rev Genet. 2011 Jun 17;12(7):487-498.
  3. Mardis ER, Wilson RK. Cancer genome sequencing: a review. Hum Mol Genet. 2009 Oct 15;18(R2):R163-168.

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