Troubleshooting Index Dropouts or No Reads Identified for Single Sample on the MiSeq i100 Series

Background

Illumina instruments leverage Index sequences to assign reads to specific samples within a library pool, though issues with Index compatibility can lead to loss of single samples within a pool. A variety of factors including library preparation issues, secondary structure, melting temperatures, and other factors can affect Index priming and sequencing quality. This typically results in the Index sequence presenting as poly-G sequences on the MiSeq i100 Series, resulting in no reads identified for that particular sample:

Figure 1: Top Unknown Barcodes report showing Index dropout for a single sample with the Index 2 (i5) index as poly-G sequences.

Most Illumina Indexes are validated to perform as expected with Illumina sequencing platforms. For Third Party or Custom libraries, it is the end user's responsibility to screen Index combinations for compatibility with Illumina sequencing platforms.

See the following Troubleshooting steps below to investigate and resolve issues with single sample dropouts in library pools sequenced on the MiSeq i100 Series.

Troubleshooting steps

Note: Issues with Index compatibility are typically rare and most Indexes are expected to be compatible.

1. Review the Top Unknown Barcodes to identify if at least one of the Indexes for the affected sample sequenced successfully. For example, in Figure 1 above, the highlighted box shows Index 1 sequenced correctly though Index 2 presents as poly-G sequences.

   1. If neither barcode is present, the issue is likely not due to Index incompatibility. Contact Illumina Tech Support for further assistance.

   2. If only one of the Indexes was affected for that sample, proceed to Step 2 below.

2. If custom Index Primers were used, review Knowledge Article Considerations when migrating non-Illumina libraries between sequencing platforms to confirm that primer Tm is compatible with the MiSeq i100 Series.

3. For custom libraries, review library design with Tech Support - Library Preparation team to confirm expected compatibility with Illumina sequencing platforms.

4. If no issues are identified with the library design or custom primers, re-sequence libraries in Read First mode.

   1. On the Review Run page during run setup, de-select the checkbox for Sequence Indexes First to select Read First sequencing (Figure 2)

5. If issue persists in both Index First and Read First sequencing modes, the Index is likely incompatible with the MiSeq i100 sequencing platform. Consider re-preparing sample using a different Index combination.

Figure 2: Review run page during run setup, highlighting the checkbox to toggle between Index First and Read First sequencing.

For any feedback or questions regarding this article (Illumina Knowledge Article #9400), contact Illumina Technical Support [e-mail].