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 labs offer this service. MR DNA Lab has 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 .

MicroRNA (miRNAs) detection using next generation sequencing (NGS) technologies

MicroRNAs are a class of short, non-coding, single-stranded RNAs (around 21–25 nucleotides in length) that act as post-transcriptional regulators in gene expression (Bartel 2009). It predominantly acts by binding to the 3′UTR of target mRNAs in the form of ribonucleoprotein complexes mediating mRNA destabilization and thereby translational repression (Krol et al., 2010). MicroRNAs are expressed in tissue, cell-type and developmental-stage-specific patterns with diverse regulatory function. Several human diseases reported to be associated with dysregulation or mutation of micro RNA genes (Zhang B, Farwell MA 2008). Besides, microRNAs have been implicated in diverse human/plant physiological process. Expression profiles of microRNAs have immense potential as diagnostic biomarkers for human diseases. Their tissue specific profiles can be identified in blood plasma, cerebrospinal fluid and urine. Reliable and efficient methods (e.g., sequencing based approaches) have been developed in order to assess microRNA profile or to discover novel microRNA in multiple biological or clinical samples (Hafner et al., 2008; Chiang et al., 2010). Next generation sequencing technology is an elegant approach to develop micro RNA biomarker discovery specific to cancer (Jima et al., 2010; Farazi et al., 2011), cardiovascular (van Empel et al., 2012) or neurodegenerative diseases (Cheng et al., 2013).  Mature microRNAs are characterized by 5′-phosphate (p) and 3′-hydroxyl (OH) groups and are highly conserved between species and most of them have been discovered and profiled in plants, humans and other mammalian species (Brown et al., 2013); however, less abundant and/or cell type-specific microRNAs remain need to be characterized. By taking advantage of the chemical properties of microRNAs protocols have been developed to enrich for microRNAs over RNA turnover (Lau et al., 2011) and generate sequencing library for next generation sequencing platforms (Hafner et al., 2011). We at MRDNA use Illumina TruSeq kit protocol to prepare microRNA sequencing libraries. Illumina micro RNA adapters are designed and optimized in such a way that they can directly, and specifically, be ligated to microRNAs. The library preparation protocol is simple and easy, includes adapter ligation, reverse transcription, PCR amplification, and pooled gel purification to generate a library product (Luo, 2012). The RNA 3′ adapter that specifically target microRNAs and other small RNAs are ligated to each end of the RNA molecule and reverse transcribed to generate cDNA. PCR amplification of cDNA using a common primer and a primer containing index sequences (Hafner et al., 2011) finally generates the sequencing library (Fig-1). With greater sensitivity this method provides the most accurate detection and quantification of rare microRNA sequences. NGS has been instrumental in the discovery and profiling of microRNAs and other non-coding RNA on any organism, without prior genome annotation. The available microRNA databases are an important resource for investigators interested in microRNA biology, diagnostics, and therapeutics (Brown et al., 2013). Whether you are searching for a novel microRNA associated with cancer, cardiovascular, renal disease, or neurological disorders or plant development we are here to help you with sequencing solutions. MR DNA has a full range of state-of-the-art equipment for next generation sequencing including Illumina, ion Torrent and 454 GS. MRDNA lab also provides data analysis and management tools for next generation sequencing solutions.

References:

  1. Bartel DP. MicroRNAs: Target Recognition and Regulatory Functions. Cell. 2009; 136:215-233.
  2. Krol J, Loedige I, Filipowicz W. The widespread regulation of microRNA biogenesis, function, and decay. Nat. Rev. Genet. 2010; 11 597-610. Zhang B, Farwell MA. MicroRNAs: a new emerging class of players for disease diagnostics and gene therapy. J Cell Mol Med. 2008; 12: 3-21.
  3. Hafner M, Landgraf P, Ludwig J, Rice A, Ojo T, Lin C, et al. Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods. 2008; 44:3-12.
  4. Chiang HR, Schoenfeld LW, Ruby JG, Auyeung VC, Spies N, Baek D, et al. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev. 2010; 24:992-1009.
  5. Jima DD, Zhang J, Jacobs C, Richards KL, Dunphy CH, et al. Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs. Blood. 2010; 116:118-127.
  6. van Empel VP, De Windt LJ, da Costa Martins PA. Circulating miRNAs: reflecting or affecting cardiovascular disease? Curr Hypertens Rep. 2012; 14(6):498-509.
  7. Cheng L, Quek CY, Sun X, Bellingham SA, Hill AF. The detection of microRNA associated with Alzheimer’s disease in biological fluids using next-generation sequencing technologies. Front Genet. 2013; 4:150.
  8. Farazi TA, Horlings HM, Ten Hoeve JJ, Mihailovic A, Halfwerk H, et al. MicroRNA sequence and expression analysis in breast tumors by deep sequencing. Cancer Res. 2011; 71:4443-4453.
  9. Brown M, Suryawanshi H, Hafner M, Farazi TA, Tuschl T. Mammalian miRNA curation through next-generation sequencing. Front Genet. 2013; 4,145,1-8.
  10. Lau N. C., Lim L. P., Weinstein E. G., Bartel D. P. () An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science. 2001; 294, 858-862.
  11. Hafner M, Renwick N, Brown M, Mihailovic A, Holoch D, Lin C, et al. () RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. RNA. 2011; 17, 1697-1712.
  12. Luo S. MicroRNA expression analysis using the Illumina microRNA-Seq Platform. Methods Mol Biol. 2012; 822,183-188.

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Microsatellite identification at MR DNA

Microsatellites, or simple sequence repeats (SSRs) are regions of DNA that contain short tandem repeats (STRs) of 1 to 6 nucleotides. Microsatellites occur ubiquitously in all prokaryotic and eukaryotic genomes (Buschiazzo and Gemmell, 2006; Kelkar et al., 2008) and are popular markers for population genetics (Guichoux et al., 2011). Microsatellite markers are one of the most informative and versatile DNA-based markers used in genetic research, however, their development has traditionally been a costly process. Recent advances in next generation sequencing technologies allow the efficient identification of large numbers of microsatellites (Hudson, 2008; Morozova and Marra, 2008) at a relatively low cost and effort of traditional approaches. NGS method produce large amount of sequence data and are used to isolate and develop numerous genome wide and gene based microsatellite loci. We at MRDNA use Illumina MiSeq platform for microsatellite identification. Sequencing and microsatellite identification steps (figure1) includes isolation and purification of genomic DNA, fragmentation, ligation to sequencing adapters and purification following the standard protocol of the Illumina TruSeq DNA Library Kit. Following the denaturation and amplifications steps libraries can be pooled and sequenced. The resulting reads are analyzed with the program PAL_FINDER_v0.02.03 (Castoe et al., 2012) to extract those reads that contain perfect 2mer, 3mer, 4mer, 5mer, and 6mer tandem SSRs. Reads are identified as SSRs if they contain simple repeats of at least 12 bp in length for 2–4mers (e.g., 6 tandem repeats for dinucleotides), and at least 3 repeats for 5mers or 6mers. The reads are then sorted by the monomer sequence of the repeat (e.g., TAC or TA repeats) and by the number of tandemly repeated units. The program is operated using a control file that determines parameter settings. A number of recent studies demonstrate the efficient use of Illumina technologies for the discovery of microsatellites in various organisms (Zalapa et al., 2012; Nunziata et al., 2012; Castoe et al., 2012).We at MR DNA routinely perform DNA sequencing and microsatellite identification and provide cost effective high quality data and robust output from only little amount of input DNA.

References:

  1. Buschiazzo, E. and Gemmell, N.J. (2006) The rise, fall and renaissance of microsatellites in eukaryotic genomes. Bioessays, 28, 1040-1050.
  2. Kelkar YD, Tyekucheva S, Chiaromonte F, Makova KD. The genome-wide determinants of human and chimpanzee microsatellite evolution. Genome Res. 2008 Jan;18(1):30-38.
  3. Guichoux, E., Lagache, L., Wagner, S., et al. (2011) Current trends in microsatellite genotyping. Molecular Ecology Resources, 11, 591-611.
  4. Hudson, M.E. (2008) Sequencing breakthroughs for genomic ecology and evolutionary biology. Molecular Ecology Notes, 8, 3-17.
  5. Morozova, O. and Marra, M.A. (2008) Applications of next- generation sequencing technologies in functional genomics. Genomics, 92, 255-264.
  6. Castoe TA, Poole AW, de Koning AP, Jones KL, Tomback DF, Oyler-McCance SJ, Fike JA, Lance SL, Streicher JW, Smith EN, Pollock DD. Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake. PLoS One. 2012;7(2):e30953.
  7. Zalapa JE, Cuevas H, Zhu H, Steffan S, Senalik D, Zeldin E, McCown B, Harbut R, Simon P. Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. Am J Bot. 2012 Feb;99(2):193-208.
  8. Nunziata SO, Karron JD, Mitchell RJ, Lance SL, Jones KL, Trapnell DW. Characterization of 42 polymorphic microsatellite loci in Mimulus ringens (Phrymaceae) using Illumina sequencing. Am J Bot. 2012 Dec;99(12):e477-480.

 

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