Prebiotics and Pasture Raised Chicken

egg-1175620_1280There seems to be a trend amongst millennials today that has its roots in a simpler time, the need for “happy” food. What do I mean by “happy” food? Well, food that is harvested from grass-fed cows and free-range chickens, food that is organic, food that is hormone free, and food that is harvested from animals that lived a “happy” life. However, happiness comes at a cost. Unless you’re one of the homesteading types, then you have most likely taken trip in the past week to pick up a carton of eggs. As you may have noticed there is a substantial price difference in a dozen conventional eggs and a dozen free-range or cage-free eggs. There is a reason for this, a team of researchers from the University of Arkansas explain that these non conventional farming methods result in a greater prevalence of foodborne pathogens and diminished health status. Essentially, choosing to raise grass-fed or free-range animals is comes at a risk to the farmer, a risk of losing more animals to disease as well as a lesser yield in comparison to conventional farming methods. Naturally, farmers also have to eat, and in order to recoup the cost of these risks, we the consumer are forced to pay a premium in order to eat as if we were on the Oregon Trail.

Park et al. recently published a study in Plos One examining the effects of prebiotics on the microbiome of free-range chickens. With the hopes of limiting the prevalence of pathogenic bacteria, feed additives, such as prebiotics, may be able to reduce the cost to farmer and consumers alike by increasing the yield and overall health of the animals. In order to examine the gut microbiota of these naked neck chickens, Park et al. decided to take advantage of next generation sequencing and perform 16s rRNA gene sequencing to complete this phylogenetic study. 16s rRNA sequencing and the microbiome have been the focus of several human studies, and as the sequencing technology continues to advance, it is exciting to see 16s rRNA gene sequencing be taken advantage of in such a capacity. DNA was extracted from the ceca of 45 birds assigned to three different groups and the V4 region of the 16s rRNA gene sequenced using the Illumina MiSeq technology. As a result, Park et al. were able to analyze gastrointestinal microbiota of the three groups and determine that “there was a significant increase in genus Faecalibacterium” which is known to be related to increased health of the host. 

 

Microbiome of Breast Milk

newborn-659685_1920Researchers from the University of Western Ontario sought out to determine how the various factors that take place during birth affect the microbiome of human milk e.g. delivery vaginally or caesarean section, gender of the infant, delivery at term or preterm. Urbaniak et al. collected milk from 39 Caucasian Canadian women for DNA isolation and 16s rRNA sequencing. The microbial data for each sample was generated using the Illumina MiSeq platform.

There is a gaining interest in the correlation between the microbiome of infants and the onset of certain diseases and/or health issues such as asthma, types I and II diabetes, and obesity. It is well known that newborns receive a boost in their immune system from breast feeding, but how the microbiome is affected by various stages of gestation is still unknown.

This study employed the use of barcoded primers to sequence the V6 hypervariable region and was able to determine that despite the differing physiological and hormonal factors between the 39 women there was no alteration to the bacterial composition found in the breast milk.

Whole-Transcriptome Sequencing (RNA-Seq)

ARNm-Rasmol.gif (353×423)Whole transcriptome analysis is important to understand genome-wide differences in RNA expression which allows Scientists to understand how altered genetic variants changes in gene expression. RNA-seq method can help to sequence different types of RNAs such as total RNA, small RNA (miRNA, tRNA and rRNA). The RNA-seq can contribute to understand the complexity of diseases such as cancer, diabetes, and heart disease.  MR DNA provides low cost Whole-Transcriptome Sequencing service to Scientists around the world. Scientists at MR DNA routinely carry out the Whole-Transcriptome sequencing using Illumina (HiSeq or MiSeq), 454 and Ion Torrent platforms. For more information please visit www.mrdnalab.com.

 

Next Generation Sequencing | MR DNA

MR DNA can perform whole-genome or de novo sequencing, resequencing … in one flow cell lane, reducing the cost of this service for small genomic libraries. …transcriptome analysis using mRNA sequencing on the HiSeq platform.

Price List | MR DNA

TruSeq RNA/small RNA sample library prep, per sample … Illumina HiSeq 2000/2500sequencing (high-output mode) 7 lanes run with samples, 1 phiX lane

 

Next Generation Sequencing Facility – MR DNA

The DNA Sequencing Facility offers all-inclusive next generation sequencing, DNA extraction and SNP genotyping services on a … We work with our customers to prepare DNA, RNA, ChIP, GBS, metagenomic, exome, and other sequencing … Run Charges – HiSeq 2000/2500 High Throughput  …

RNA-Seq (Quantification) | MR DNA

Powered by our unique bioinformatics capabilities, MR DNA RNASeq services deliver …RNASeq based on Hiseq: high-throughput and a wide  …

 

Sequencing – Genome Technology | MR DNA

https://mrdnalab.com

… instructions. Below you will find more information about our sequencing services: library preparation, sequencing, and analyses. … mRNASeq · ChIP-Seq …Sequencing. GTAC operates four Illumina HiSeq 2500 sequencing instruments.

 

MR DNA: HiSeq 2500 & MiSeq – Next Generation Sequencing

The MR DNA Facility houses an Illumina HiSeq 2500 and an … such as ChIP-Seq, transcript counts, SNP detection, and small RNA analysis, the … the mate-pair protocol and should contact the facility regarding this service.

 

ChIP-Seq (Chromation immunoprecipitation sequencing) at MRDNA

truseq-chip09242013.png (960×720)Chromatin immunoprecipitation (ChIP) is a powerful method for studying interactions between specific proteins and a genomic DNA region. MRDNA routinely performs ChIP-seq and provide cost effective high quality data, global binding site maps for a protein of interest and robust output. Please visit www.mrdnalab.com.

Our services – MR DNA Laboratory

MR DNA

Illumina HiSeq 2500/200, MiSeq – The HiSeq 2500/2000 sequencing systems offer the … Metagenomics and amplicon sequencing; ChIPSeq  …

 

Functional Genomics

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MR DNA offers library prep, sequencing, and basic data analysis services for …Simple ChIPSeq … hiSeq2500, …., sequence the samples

 

High-Throughput Sequencing Center

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We provide services for high-throughput next-generation sequencing using the … (ChIPSEQ), RNA discovery and Multiplex sequencing … Pricing forHiSeq 2500 sequencing is based on ……..sequencing run,  …

 

MR DNA Research Laboratory Services

Services. Transcriptomics. mRNA-Seq: Stranded and non-stranded, high levels of multiplexing … ChIPSeq. Transcription factor analysis; Histone modifications

NGS with longer read length and better quality at MR DNA

Next generation sequencing (NGS) has been instrumental in advancing scientific fields from human disease research to environmental and evolutionary science. MR DNA is your next generation sequencing and bioinformatics service provider and collaborator. We at MR DNA routinely perform next generation sequencing on Illumina platform. Recently Illumina has launched new reagent kits for the MiSeq system to double sequencing output. translations Now the output is increased to 15 GB with new reagent kits and a single run generates upto 50 million sequencing reads, each 300 bases long. Data quality is even more robust with greater than 70% of bases above Q30 at 2×300 base-pairs. This development helps in improving the quality of the genome assemblies and advancing applications that require longer read lengths for the projects such as cancer, genetic disease, microbiology, agriculture and forensics. We have successfully performed several runs using 2X300 Miseq reagent kit and we are here to help you with sequencing solutions.

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 resequencing 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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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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Sequencing low diversity libraries at MR DNA

Next generation sequencing (NGS) has been instrumental in advancing scientific fields from human disease research to environmental and evolutionary science. It has been used to investigate sample diversity in amplicon libraries and has been instrumental in determining microbial diversity. We at MRDNA use Illumina MiSeq platform for 16S sequencing and analysis. With new version of Illumina MiSeq Control Software (MCS) and inclusion of 5% PhiX significantly generate improved quality data for low-diversity samples. We have successfully performed several very low diversity runs at our lab with mostly 16S samples. This is a promising platform for researchers wishing to do 16S sequencing & analysis and have large number of samples. We at MR DNA routinely perform 16S sequencing with large volume of samples and provide cost effective high quality data and robust output from only low input DNA. Whether you are performing metagenomics studies, or monitoring disease outbreaks, we are here to help you with sequencing solutions.

Metagenome Sequencing at MR DNA

Next generation sequencing (NGS) has been instrumental in advancing scientific fields from human disease research to environmental and evolutionary science. Genetic material recovered directly from environmental samples is termed as metagenomes. Metagenomics is defined as the direct genetic analysis of genomes contained with an environmental sample (Thomas et al., 2012). In other words, metagenomics refers to the study of genomic DNA obtained from microorganisms that cannot be cultured in the laboratory (Streit and Schmitz, 2004). This represents the vast majority of microorganisms on earth (Bik et al., 2012). Metagenomics provides access to the functional gene composition of microbial communities and thus gives a much broader description than phylogenetic surveys, which are often based only on the diversity of one gene (e.g., 16S rRNA gene). Metagenomics is also a powerful tool for generating novel hypotheses of microbial function; the discoveries of proteorhodopsin-based photoheterotrophy or ammonia-oxidizing Archaea are the examples (Eisen, 2007; Marco, 2011; Beja et al., 2000; Nicol et al., 2006). With NGS, it is possible to measure changes anywhere in the genome without prior knowledge. Single-base resolution allows tracking of microbial adaptation over short periods of time, both in the laboratory and in the environment. NGS provides comprehensive solutions for studying microorganism on microbial genome level and metagenome level. A sequence-based metagenome project involves several steps (Fig-1). Metagenome sequencing steps includes isolation and purification of genomic DNA, fragmentation, ligation to sequencing adapters and purification. Barcoded indices are added during the amplification steps.  Following the denaturation and amplifications steps libraries can be pooled and sequenced. We at MRDNA use Nextera DNA sample preparation kits (illumina) for metagenome sequencing library preparation. In short, this Kit uses an engineered transposome to simultaneously fragment and tag (“tagment”) input DNA, adding unique adapter sequences in the process. A limited-cycle PCR reaction uses these adapter sequences to amplify the insert DNA. The PCR reaction also adds index sequences on both ends of the DNA, thus enabling dual-indexed sequencing of pooled libraries on any Illumina Sequencing System (Fig-2). MR DNA offers metagenomics services with the MiSeq. With an unprecedented ability to multiplex, the MiSeq system and its enhanced workflow, provides the most comprehensive and cost effective solutions for all metagenomic studies. We at MR DNA routinely perform metagenome sequencing with large volume of samples and provide cost effective high quality data and robust output from only 50 ng of input DNA. Whether you are performing metagenomics studies, or monitoring disease outbreaks, we are here to help you with sequencing solutions.

References:

  1. Thomas T, Gilbert J, Meyer F. Metagenomics – a guide from sampling to data analysis.Microb Inform Exp. 2012 Feb 9;2(1):3.
  2. Streit WR, Schmitz RA. Metagenomics–the key to the uncultured microbes. Curr Opin Microbiol. 2004 Oct;7(5):492-8.
  3. Bik, H. M., Porazinska D. L., Creer S., Caporaso J. G., Knight R., et al. (2012) Sequencing our way towards understanding global eukaryotic biodiversity. Trends Ecol Evol 27: 233–243.
  4. Eisen, JA. Environmental Shotgun Sequencing: Its Potential and Challenges for Studying the Hidden World of Microbes”. PLoS Biology 2007;5 (3): e82.
  5. Marco, D. Metagenomics: Current Innovations and Future Trends. Caister Academic Press. 2011; ISBN 978-1-904455-87-5.
  6. Beja O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP, Jovanovich SB, Gates CM, Feldman RA, Spudich JL, Spudich EN, DeLong EF: Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 2000, 289(5486):1902-1906.
  7. Nicol GW, Schleper C: Ammonia-oxidising Crenarchaeota: important players in the nitrogen cycle? Trends Microbiol 2006, 14(5):207-212.

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Exom sequencing

Exome sequencing is a strategy to selectively sequence the coding regions of the genome. Exons are short, functionally important sequences of DNA which represent the regions in genes that are translated into protein. This also includes untranslated regions (UTRs) however, are usually not included in exome studies. Exomes are ideal to help us understand high-penetrance allelic variation and its relationship to phenotype. In the human genome, exons constitute about 1% of its genome (Ng et al., 2009) and that the protein coding regions of the human genome constitute about 85% of the disease-causing mutations (Choi et al., 2009). The available strategy for exom sequencing mainly focuses on generating reads protein coding regions of the genome by using high-throughput sequencing technologies after exome enrichment steps. Exome sequencing steps includes random sharing of genomic DNA to construct the library by ligating adaptors. The library is enriched for sequences corresponding to exons by aqueous-phase hybridization capture. In this step the fragments are hybridized to biotinylated DNA baits in the presence of oligonucleotides that blocks the adaptors. Recovery of the hybridized fragments by biotin–streptavidin-based pulldown is followed by amplification and massively parallel sequencing of the enriched, amplified library and the mapping and calling of candidate causal variants (Fig-1). For sample indexing barcodes can be introduced during the initial library construction or during post-capture amplification. The critical parameters in the process include the degree of enrichment, the uniformity with which targets are captured and the molecular complexity of the enriched library (Bamshad et al., 2011). We at MR DNA use TruSeq exome enrichment kit (Illumina) for the sample library preparation. It features a highly optimized probe set that delivers comprehensive coverage of exomic sequence starting from only 1ug of DNA input. This kit include >340,000 95mer probes, each constructed against the human NCBI37/hg19 reference genome. The probe set was designed to enrich >200,000 exons, spanning 20,794 genes of interest. Each 95 mer probe targets libraries of 300-400 bp, enriching 265- 465 bases centered symmetrically around the midpoint of the probe. Sequence data generated from exome enrichment are analyzed using a script to generate two sets of statistics; post alignment and post CASAVA (Consensus Assessment of Sequence and Variation) analysis. Data can be visualized to examine the on- target and off-target coverage in a sample using GenomeStudio Data Analysis Software. With the combination of next generation sequencing technologies and robust and efficient methods of sequence capture (Hodges et al., 2007), exome sequencing has become a useful tool for the discovery of genes underlying rare monogenic disease, and the discovery of coding variants associated with common disease (Ng et al., 2009, 2010; Choi et al., 2009; Singleton, 2011). We at MR DNA routinely perform exom sequencing and provide cost effective high quality data and robust output from only 1 ug of input DNA.

References:

  1. Ng SB, Turner EH, Robertson PD, et al: Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009; 461: 272-276.
  2. Choi M, Scholl UI, Ji W, et al: Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci U S A 2009 106 (45): 19096–19101.
  3. Bamshad MJ, Ng SB, Bigham AW, Tabor et al: Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 2011 Sep 27;12(11):745-55.
  4. Ng SB, Buckingham KJ, Lee C et al: Exome sequencing identifies the cause of a mendelian disorder. Nat Genet. 2010; 42: 30-35.
  5. Hodges E, Xuan Z, Balija V et al: Genome-wide in situ exon capture for selective resequencing. Nat Genet. 2007; 39: 1522-1527.
  6. Singleton AB. Exome sequencing: a transformative technology. Lancet Neurol. 2011 Oct;10(10):942-6.

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