Questions & Answers

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  • Libraries


  • This depends on the size of the organism you are trying to resequence. For whole-genome resequencing, a 25-fold over-sampling should be adequate. For targeted resequencing involving mixes of many PCR products, 75-fold over-sampling will correct for the inability to mix the PCR products at a 1:1 ratio. Illumina sample prep shows no systematic bias. In sequencing the X chromosome, we achieved 16-fold average cover­age with all sequenceable bases covered at least twice.
    There are no inherent limits in the software. Illumina scientists have pooled 29 BACs of 130 kb each.
    Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.

    The HiSeq v4 reagent kits support dual-indexing workflows without requiring the purchase of additional SBS agents. Sample prep for dual-indexed libraries requires that both indexes be present on the library. However, the second index does not need to be read during sequencing. A single-indexing workflow is supported on Illumina sequencing instruments, where only Index 1 is used. See the instrument user guide for more information about setting up an 8-base single-indexed sequencing run.

For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.

  • Analysis


  • Image analysis occurs in real time, phasing estimates and base calling start after cycle 12, and base calling and quality scoring starts after cycle 25.

    It is the ability to distinguish between two or more clusters that are in close proximity to each other.

    To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data are filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.

    Clusters passing filter are represented by PF in analysis reports. Clusters pass filter if no more than one base call in the first 25 cycles has a chastity of < 0.6.