Login

Scientific

Deep Sequencing Notes

These pages document notes I collected on various aspects of deep sequenmcing, aka sequencing by synthesis or short fragment sequencing. The machinie on which my experience is based is a Illumina/Solexa technologies Genome Analyzer II. The work I performed was related to the deep sequencing facility in Basel (at ETH Zurich) and these are some extra-curricular notes that remain otherwise unused.

We also brought the illumina documentation for version 0.3 and 1.0 of the pipeline online for users intersted in understanding the working of the pipeline in detail. We collected some notes on compiling the pipeline

One of the initial aspects of Deep Sequencing is the question whether there is a bias present and what types of biases are known.

  • One of the surprising biases we learned in Lausanne is the fact that higher cycles will favour specific bases. This is shown in detail here.
  • Another important bias is temperature dependence.
  • In one of our experiments we essentially sequenced the adaptor which was nicely visible in the IVC (Intesnity versus Cycle) plot.

    • One of the biggest problems with the software associated with the genome analyzer is that it relies on the known genome to decide on the quality of the reads. This process will even modify the quality values of the different reads. This then leads to a number of quality reports based on the alignemnts. We present two examples here. The first quality report is based on the eland aligner (quality report). The second quality report is based on the phagealign aligner. The output of the GA Pipiline is discussed in detail at the file formats page.

    Illumina software

  • What with more than 16M tags ? An important problem we had was that the eland aligner was not able to handle more than 16'000'000 tags. This document explains how to resolve this.
  • A race condition in the goat pipeline.
  • How to split lanes ? - explains how to split the output of the analysis pipeline such that we can give each custumer their own quality report.

    • Our own software

  • Eland2Wig - An eland2 to wig file convertor
  • Eland2Exp - An eland2 to expression file convertor. This converts your genome analyzer into a microarray scanner.
  • Sig22Seq - A tool to convert the *.sig2.txt files to filtered sequences.
  • ProbPerSf - A tool to shorten the measured fragments up to a length that can be trusted.
  • Sample Tracking - A rudimentary sample tracking system created to keep track of incoming samples and label them appropriatelly.
  • Mailing list - how to use the software and our experience with the GA2 Genome Analyzer.

    • More Deep Sequencing notes
    Web: http://analysis.yellowcouch.org/
    Mail: werner@yellowcouch.org
    http://werner.yellowouch.org/