Questions & Answers

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General

  • How can I create reports of ForenSeq Universal Analysis Software data?

    To create a report for all the analysis results in a project, or for a sample result, a report can be generated from the project page.  A report for a sample result can also be generated from the sample details page. After a report is created, it can always be retrieved again from the project dashboard.

  • How do I determine if the positive and negative controls have passed in a MiSeq FGx run?

    The software provides a pass or fail indication for each positive and negative control sample. The pass/fail indication is based on the expected performance of the control.

  • What QC indicators are included in ForenSeq Universal Analysis Software?

    For STR typing, ForenSeq Universal Analysis Software presents a locus quality control indicator for the following conditions:

    - More alleles are present than expected

    - Alleles are not balanced

    - An allele is below the interpretation threshold but above the analytical threshold

    - The number of reads was not enough to make a call

    - Stutter is present above the allowable level for the locus

     

    For SNP typing, ForenSeq Universal Analysis Software presents a locus quality control indicator for the following conditions:

    - Alleles are not balanced

    - An allele is below the interpretation threshold, but above the analytical threshold

    - The number of reads was not enough to make a call

  • How is the Illumina technology different from the CE-based technology for ForenSeq DNA Signature Prep?

    Where CE-based assays give the length of the STRs, Illumina technology provides, not only the length of the STRs, but any variation in the STRs and genotypes at the various SNPs. Illumina technology also allows multiple samples to be pooled together and sequenced in one run, instead of running samples one at a time.

  • What does ForenSeq Universal Analysis Software do?

    The ForenSeq Universal Analysis Software is used to create runs, monitor runs, review results, and align reads to make allele and genotype calls for sample information. Optionally, the software can generate reports, population statistics, sample comparisons, phenotype estimations and bio-geographical ancestry estimations.

  • What is a cluster passing filter and how does it affect MiSeq FGx run data?

    Clusters passing filters are those clusters that pass a number of sequencing quality thresholds. % passing filter is calculated over the first 25 cycles of sequencing, and only clusters that pass filters are included in the final analysis.

  • How is the STR and SNP data displayed in ForenSeq Universal Analysis Software?

    STR and SNP data are shown in multiple formats in the ForenSeq Universal Analysis Software.  On the sample details screen, the genotype and/or warnings for each locus is displayed A detailed presentation is available for each locus by selecting it. For STR loci, a table of alleles is provided as well as a bar chart of the alleles and their relative intensities (# of reads). For SNP loci, a table of alleles is provided as well as a pie chart showing the distribution of the alleles.

  • Why is the run on the MiSeq FGx composed of two fragment reads and two index reads?

    The first read is a 351 cycle read to sequence the longer STRs present, while the 31 cycle read two acts to confirm primer pairing. The two index reads allow for the barcodes to be read, to enable deconvolution of multiple samples within a single library.

  • What is phasing and prephasing and how can this impact the quality of MiSeq FGx run data?

    Phasing and prephasing occurs when a strand within a cluster falls behind or ahead respectively of the incorporation cycle. The % metric given shows an estimate of the fraction of strands that are phased or prephased. Low percentages indicate good run statistics.

How do I know if the MiSeq FGx run met the specifications?

The run has met the Illumina specifications when the run metrics are within the thresholds and are colored green.

Workflow

  • How can I change my password? Can I retrieve or reset a forgotten password or account name?

    You need your current password to change your password. You cannot retrieve or reset a forgotten password or account name-- you must create a new account. See the ForenSeq Universal Analysis Software Guide.

     

  • Can I change the analysis after it has been completed in ForenSeq Universal Analysis Software?

    Yes, when adjusting the analysis settings from a current analysis, a new analysis is initiated. Additionally, the alteration of sample indexes or sample types automatically initiates a new analysis.

  • What data are needed for each sample to enable a run to be set up in ForenSeq Universal Analysis Software?

    For each sample, the following information is required: SampleName, SampleDescription, Project, i5Index, i7Index SampleType. See the ForenSeq Universal Analysis Software Guide for more details on these fields.

Do I have to have the server with ForenSeq Universal Analysis Software connected to the MiSeq FGx?

Yes, in order for the results to be transferred from the MiSeq FGx to the ForenSeq Universal Analysis Software a connection must exist between the systems. In many cases that connection is established through the company network. However, in some labs that connection can be on a local network created with the provided router.

Can a MiSeq FGx run be started when the server is switched off?

To log in to MiSeq FGx Control Software, a connection must be established with the ForenSeq Universal Analysis Software server to check authentication. After authentication, an RUO run can be performed without connection to the server. There is a buffering mechanism with the MiSeq FGx Control Software to allow data transmission to resume upon a connection loss to ForenSeq Universal Analsyis Software during acquisition.

Compatibility

Can the sample sheet for the MiSeq FGx run be created using the Illumina Experiment Manager (IEM)?

No. However, a sample sheet created using IEM is compatible with the RUO mode of the MiSeq FGx Control Software. For forensic runs, the samples are defined in the ForenSeq Universal Analysis Software before acquisition through a file upload or manual entry. For details on the information in the file, see the ForenSeq Universal Analsyis Software Guide.