Troubleshooting demultiplexing issues using MiSeq Reporter


The MiSeq Reporter analysis software outputs a demultiplexing summary file called DemultiplexSummaryF1L1.txt that can be used to troubleshoot demultiplexing issues.

Where is the DemultiplexSummaryF1L1.txt file?

    After FASTQ file generation completes, the DemultiplexSummaryF1L1.txt file is located in:
    <run folder>\Data\Intensities\BaseCalls\Alignment.

What information is in the DemultiplexSummaryF1L1.txt file?

    The DemultiplexSummaryF1L1.txt file has 2 sections. The top section (Figure 1) is a tab-delimited table that summarizes the demultiplexing results on a per-tile basis. On the left-hand side of the table is a list of the tiles on the flow cell. Along the top of the table, the samples are listed in the order in which they were entered into the sample sheet. Sample 0 is always reserved for the undetermined reads. The table shows the percentage of reads demultiplexed to each sample, per tile. In general, for a given sample, the percentage of demultiplexed reads should be similar across all tiles. The tile summary information can be used to identify tile-specific demultiplexing issues.

Figure 1: The tile summary section shows the percentage of reads demultiplexed to each sample, per tile.

The second section of the DemultiplexSummaryF1L1.txt file (Figure 2) lists the 100 most common index sequences (whether assigned or undetermined) for Index Read 1 and Index Read 2, and the number of times each index was found. The list also shows the reverse complement of the index sequences found (for your reference only). In the DemutiplexSummaryF1L1.txt file, periods (...) in the index sequences represent positions where the base calling software on the MiSeq could not make a base call.

Figure 2: The most popular index sequences section of the DemultiplexSummaryF1L1.txt file lists the top 100 index 1 and index 2 sequences found.

When troubleshooting demultiplexing issues, the lists of index sequences can be used to compare the expected index sequences (those in the sample sheet) to those that were found.

Some common causes for poor demultiplexing that this list can reveal are:

  • Index sequences entered in the wrong orientation in the sample sheet.
  • Incorrect index sequences entered in the sample sheet (eg, Nextera vs TruSeq HT, or index A001 vs index A006).
  • Sample mix ups between lanes.
  • Poor Index Read sequencing quality (eg, index sequences containing lots of periods, representing “no calls,” near the top of the list).