It is possible to combine digitalMLPA with other sequencing libraries in a single next-generation sequencing (NGS) run. However, certain considerations must be taken into account:
- When using standard Illumina paired-end sequencing runs, the first read must make use of the standard Illumina Rd1 primer and must have a length of at least 110 cycles.
- digitalMLPA samples should not be defined as sample in the run setup in Illumina's software.
- Optimisation may be needed. The quality checks and scores of the other libraries (performed by Illumina's software) should be considered leading. The quality of the digitalMLPA library is assessed by Coffalyser digitalMLPA.
- The amount of digitalMLPA in the combined library should not exceed 20% of the single-read capacity of the used Illumina reagent kit as this may adversely affect the quality scores calculated by the Illumina software.
Index reads of the other NGS library will not affect sequencing of the digitalMLPA library.
Sequencing mix preparation
- Prepare a digitalMLPA library according to the guidelines in the digitalMLPA General Protocol for the sequencer you are using, or contact us if no instructions for your sequencer are available. No library concentration measurement is needed. The final digitalMLPA library concentration should not be adjusted to match the concentration of other NGS libraries.
- Prepare your other sequencing library according to your standard protocol. Do not adjust the concentration of your library to match the concentration of the digitalMLPA library.
- Determine the number of required digitalMLPA reads. This should not exceed 20% of the capacity of the Illumina reagent kit used.
- The number of required digitalMLPA reads = number of samples × number of probes in the used digitalMLPA probemix × 500 (minimum read depth per probe).
- If you want to know how many digitalMLPA samples can be included, use the formula listed in this support article. Select Other as Illumina reagent kit and enter 20% of the reagent kit capacity as the reagent kit capacity.
- Calculate the percentage of total reads available for the reagent kit that will be used by digitalMLPA reads. The remainder is the percentage of total reads available for the other sequencing library.
- Combine the digitalMLPA and other sequencing libraries in proportion to the percentage of reads required for each library.
- Load the prepared mixture on the sequencer.
Suppose that SALSA® digitalMLPA™ Probemix D001 Hereditary Cancer Panel 1 version C1 is used, which contains 725 probes, 8 digitalMLPA samples need to be run, and MiSeq Reagent Kit v3 of 150 cycles (25 million single-end reads) is used.
- Calculate the number of reads available and needed for the digitalMLPA library.
- Available number of reads: 5 million (20% of 25 million).
- Required number of reads: 2.9 million (8 samples × 725 probes × 500 (minimum reads per probe)).
- As the required number of reads is lower than the available number of reads, all digitalMLPA samples can be included in the combined library. If the required number of reads is higher than the available reads, use an Illumina reagent kit with a larger capacity or include fewer digitalMLPA samples in the combined library.
- Calculate the percentage of total reads required for digitalMLPA reads.
- The required percentage digitalMLPA reads is approximately 12% (2.9 million digitalMLPA reads/25 million (Illumina MiSeq Reagent Kit v3 capacity)).
- Combine the digitalMLPA library and other sequencing library in proportion to the percentage of reads required for each library. For the MiSeq, 600 µl of the (combined) denatured library needs to be loaded for sequencing.
- 72 μl (12% of 600 μl) of the digitalMLPA library should be added to the combined sequencing library.
- 528 μl (88% of 600 μl) of the other library should be added to the combined sequencing library.
- Load the prepared mixture on the sequencer.
FASTQ file for analysis of digitalMLPA reads
Analyse the digitalMLPA data with Coffalyser digitalMLPA™. When paired-end sequencing is performed, digitalMLPA reads will be placed in an Undetermined FASTQ file. This is because digitalMLPA amplicon clusters do not generate second reads or index reads. This FASTQ file is typically named Undetermined_S0_L001_R1_001.fastq.gz (L001 = Lane, R1 = Read 1) and can be used by Coffalyser digitalMLPA for analysis.
Failed reads originating from the other sequencing library may also be included in the Undetermined FASTQ file. However, these reads will be discarded by Coffalyser digitalMLPA as they will not be recognized as valid digitalMLPA reads. Failed reads from the other library will therefore not influence digitalMLPA results.