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The genomics section offers massive sequencing service using two platforms: SOLID 5500 XL (Life Technologies) and 454 GS Junior (Roche). It offers advice on designing the project and choosing the most appropriate technology. Moreover, all the sequencing process, including the preparation of libraries, emulsion PCR and sequencing in the selected platform, is carried out in the laboratory. After obtaining the results, we provide the primary analysis of the data, and offer advanced bioinformatic analysis.

Solid 5500XL

This platform enables massive parallel sequencing of clonally amplified DNA fragments attached to beads. The sequencing methodology is based on the sequential ligation of fluorescent probes. It obtains sequences up to 75 nt. It allows to perform pair-end sequencing to obtain two sequences of each fragment, 75nt forward and 35nt reverse.

Platform characteristics

  • 99.99% average sequence accuracy.
  • Read length:
    • Single-read sequencing: 35-75 nt.
    • Pair-end sequencing: 75+35 nt.
  • Exact Call Chemistry (ECC) for single-read sequencing to obtain reads in bases.

Applications

RNA-Seq

  • Transcriptome sequencing:
    • Expression of all coding and non-coding RNAs.
    • Alternative splicing identification.
    • cSNPs identification.
    • Identification of allele-specific expression patterns.
    • Preparing libraries allows to identify the string corresponding to each sequence.
  • Small RNA sequencing: obtaining the miRNA profile of the samples and identifying new miRNA.

DNA-Seq

  • Exome sequencing:
    • Whole exome enrichment with Agilent whole exome sureselect or similar technologies.
    • Sequencing and mapping. Detection of mutations: SNPs, indels, cSNPS.
  • Whole genome resequencing:
    • Detection of mutations: SNPs, indels, cSNPS.
  • Other applications: consult with the laboratory staff.

Sample requirements

  • All the sequencing process: preparation of libraries, emulsion PCR and sequencing, performed by the laboratory staff.
  • DNA samples:
    • Provide 100 ng to 5 μg in 10-100 μl of nuclease-free water or lowTE.
    • The samples must be free of any contaminant. Perform purification using columns (Qiagen or similar), AMPure Beads (Beckman Coulter) or precipitation with ethanol. In the latter case, completely remove traces of ethanol so as not to interfere in the enzymatic steps for preparing the libraries.
    • Quantify samples accurately. It's better to use a fluorimetric method such as Quant-IT (Qubit, Invitrogen), than to do it by spectrometry.
    • Provide an image of the agarose gel of the samples to confirm their integrity. Genomic DNA must be observed as a single band >12 kb.
    • If the sample is cDNA, indicate the method used for its synthesis.
  • RNA samples:
    • The samples must be free of any contaminant. Perform purification using columns (Qiagen RNeasy, Invitrogen Ribominus or similar) or precipitation with ethanol. In the latter case, completely remove traces of ethanol so as not to interfere in the enzymatic steps for preparing the libraries. Elute or resuspend the RNA in RNase-free water.
    • Treatment with DNase must be necessary to remove traces of gDNA.
    • Quantify samples accurately. Use a fluorimetric method such as Quant-IT (Qubit, Invitrogen) or the Agilent Bioanalyzer 2100.
    • It is recommended to use total RNA samples with a value of RIN>7 to proceed with the removal of rRNA or mRNA enrichment.
    • Required amounts of RNA in RNase-free water:
RNA type Quantity required
Total RNA >10 μg in 10-20 μl
rRNA-depleted RNA >0.5 μg in 10 μl
mRNA-enriched RNA >0.5 μg in 10 μl
smallRNA consult

454 GS Junior

Servicios de secuenciación masiva

GS Junior is a "mini" version of 454 GS FLX. It uses pyrosequencing, and obtains sequences of 400nt.

Platform characteristics

  • 120,000 average reads for shotgun sequencing.
  • 90,000 average reads for amplicon sequencing.
  • Average read length: 400 bases.
  • 99% sequence accuracy.
  • Samples multiplexing capacity by use of bar codes.

Applications

  • Novo sequencing of microbial genomes, viral genomes and small genomes.
  • Amplicon sequencing (PCR product).
  • Metagenomics.
  • 16S sequencing.
  • HIV, BRCA, etc. sequencing
  • cDNA sequencing.
  • Other applications: consult with laboratory staff.

Sample requirements

  • Shot-gun or paired-end libraries:
    • The samples must be free of any contaminant. Perform purification using columns (Qiagen or similar), AMPure Beads (Beckman Coulter) or precipitation with ethanol. In the latter case, completely remove traces of ethanol so as not to interfere in the enzymatic steps for preparing the libraries.
    • Quantify samples accurately. It's better to use a fluorimetric method such as Quant-IT (Qubit, Invitrogen), than to do it by spectrometry.
    • Provide an image of the agarose gel of the samples to confirm their integrity. Genomic DNA must be observed as a single band >12 kb.
Library type Quantity of DNA required
Fragments 3 μg in 100 μl
Paired-end 3 kb 10 μg in 100 μl
Paired-end 8 kb 20 μg in 100 μl
Paired-end 20 kb 50 μg in 100 μl
  • If the sample is cDNA, indicate the method used for its synthesis.
  • Amplicons libraries:
    • PCR conditions have to be optimised to ensure the amplification of a single fragment of the desired size. Otherwise, it is recommended to select the size of the desired amplicon on an agarose gel or similar methodology.
    • The samples must be free of any contaminant. Perform purification using columns (Qiagen or similar), AMPure Beads (Beckman Coulter). Perform elution in nuclease-free water.
    • After purification, evaluate the quality of the samples on a 2% agarose gel. Include an image of the gel when sending the samples.
    • Quantify samples accurately. It's better to use a fluorimetric method such as Quant-IT (Qubit, Invitrogen), than to do it by spectrometry.
    • Provide 100 ng of amplified DNA in 10-20 μl of water for each sample.
    • We ned information on the design of the amplicon: indicate for each sample the complete sequence of the forward and reverse primers including adapter, MID and specific primer of the amplicon.
 
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