Troubleshooting

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Plan

What is the best library prep kit for my application?

Determine the best kit for your needs based on your project type, starting material, and method of interest using the Library Prep and Array Kit Selector tool.

My coverage was insufficient per sample

Illumina provides an online Sequencing Coverage Calculator tool that calculates the reagents and sequencing runs needed to arrive at the desired coverage for your experiment, based on the Lander/Waterman equation.

For more information about calculating coverage estimates, see the technical note Estimating Sequencing Coverage.

My custom libraries did not cluster

There are several possible reasons why your custom libraries did not cluster. For more information on the indexing strategies for each instrument, refer to the Indexed Sequencing Guide.

  1. Incompatible adapter sequences. You can research appropriate sequences with the Illumina Adapter Sequences document. This document provides the nucleotide sequences that comprise Illumina oligonucleotides used in Illumina sequencing technologies. These sequences are provided for the sole purpose of understanding and publishing the results of your sequencing experiments.
  2. The primers were not in the appropriate wells. Refer to the MiSeq System Custom Primers Guide. You may also want to spike in your custom primers. For more information, see the bulletin Spiking custom primers into the Illumina sequencing primers.
  3. All libraries prepared with the current Illumina library preparation kits are compatible with all Illumina sequencing platforms. Sequencing libraries prepared with non-Illumina library preparation methods may require additional optimization on different sequencing platforms. For more information, see the bulletin Considerations when migrating non-Illumina libraries between sequencing platforms.

Can I use custom primers on the MiSeq?

Refer to the MiSeq System Custom Primers Guide.

You may also want to spike in your custom primers. For more information, see the bulletin Spiking custom primers into the Illumina sequencing primers.

What is the typical MiSeq output run folder size?

For more information, see the bulletin Approximate sizes of sequencing run output folders.

Prep

Achieving consistent sample to sample normalization

For recommendations to improve sample to sample normalization, see the bulletin Best practices for manually normalizing library concentrations.

PhiX spike-in and low diversity library preparation considerations

Nucleotide diversity is required for effective template generation on Illumina sequencing platforms and is important for the generation of high-quality data. High nucleotide diversity is when a library has roughly equal proportions of all 4 nucleotides in every cycle of the run. Use the following resources to understand and more effectively sequence low-diversity libraries.

How do I convert ng/ul concentrations to nM?

Illumina libraries, such as Nextera, require the use of dsDNA-specific fluorescent dye methods for accurate quantification. These methods typically measure dsDNA concentration in ng/µl. For instructions, see Converting ng/uL to nM when calculating dsDNA library concentration.

Library quality control and consistent quantification

Why do variations and inconsistencies arise in quantitation for next-generation sequencing? Illumina provides several resources and recommendations for quantitation methods.

Run

MiSeq System Checks

An Illumina Support Representative may request you perform a system check on the MiSeq during the course of investigating a performance issue. A system check is a series of diagnostic tests to identify potential hardware issues with the system.  For more information on the System Checks and how to set up a system check, review the video How to Perform a MiSeq System Check.

MiSeq fails to recognize reagent RFID

MiSeq reagents are equipped with a radio-frequency identification (RFID) tag to enable accurate consumable tracking. If the RFID fails to be detected, a run can be started using a bypass code created by the MiSeq Self-Service tool available through your MyIllumina account. For more information, see the bulletin How to generate RFID bypass codes for the MiSeq.

How do I set up a PhiX only validation run?

A PhiX validation run confirms proper hardware and software performance of the instrument.  Instructions for setting up the sample sheet for a PhiX validation run for the MiSeq system using Illumina Experiment Manager (IEM) can be found in the bulletin How to set-up the sample sheet for a PhiX validation run on the MiSeq system using Illumina Experiment Manager?

It is important that a PhiX control run fall within optimal cluster densities for each platform, as listed in the bulletin PhiX loading concentrations for validation runs on Illumina sequencing platforms

The PhiX Control v3 Library (commonly referred to as PhiX, FC-110-3001) is derived from the small, well characterized bacteriophage PhiX genome. For more information on this tool, see the bulletin What is the PhiX Control v3 Library and what is its function in Illumina Next Generation Sequencing?

Pre-run flow check errors

If the MiSeq fails to measure flow rate when starting a run, please see the bulletin What should I do if my MiSeq fails to measure flow rate when starting a run?

Best focus or no usable signal errors

“Best focus not found,” “Best focus is too near the edge of range,” or “No usable signal found, it is possible clustering has failed,” errors that occur after the first cycle of imaging on the MiSeq indicate there may be insufficient cluster intensity for the instrument to find the best plane of focus. For more information, see the bulletin How to troubleshoot the best focus and no usable signal errors on the MiSeq.

Restarting Real Time Analysis (RTA)

RTA (Real-Time Analysis) is an integrated software that analyzes flow cell images and calculates metrics during a run. You may need to manually stop and restart RTA if it is stalled or if the network path is down. For more information, see the bulletin How to restart RTA on MiSeq system.

Cluster density optimization

Cluster density is a critically important metric that influences run quality, reads passing filter, Q30 scores, and total data output. For more information, see the following resources:

Can I sequence less than 25 cycle in Read 1?

If specified during run setup, Illumina sequencing systems can perform fewer than 25 cycles for Read 1. However, this option is not recommended nor supported. For more information, see the bulletin Why sequencing 25 or more cycles in Read 1 is recommended.

Run performance: Does my run look good?

How do you assess your runs performance? Illumina has several tools to help you with this, including the Sequencing Analysis Viewer (SAV) Software. See the following resources for more information.

Where can I find lot specific information about my sequencing and library prep consumables?

The Product Lot Tracker allows you to:

  • Find and download a Certificate of Conformance (C of C)
  • Look up expiration dates
  • Use the Serial Number to view all items in your kit box

How do I properly dispose of the sequencing waste?

The Illumina Internet Safety Data Sheet (SDS) search page is a search engine that displays published Illumina GHS compliant SDSs. The SDS search page allows for the entry of search criteria to locate and view a required SDS. 

The following bulletin may also be useful: What is the final formamide concentration in the waste solution of Illumina sequencing systems?

Analyze

I have questions related to the MiSeq Reporter software

The MiSeq Reporter analysis software processes base calls generated on-instrument during the sequencing run by Real Time Analysis (RTA) software, and produces information about alignment, structural variants, and contig assemblies for each genome requested and each sample based on the analysis workflow specified in the sample sheet.

I have questions related to the Basespace Sequence Hub (BSSH)

BaseSpace Sequence Hub is the Illumina cloud computing environment for analyzing sequencing data. You can stream data directly from your sequencing run from HiSeq, NextSeq, MiSeq, and MiniSeq systems.