Input recommendations FAQ for the TruPath Genome assay

Which species are supported for TruPath sequencing (eg, only human? Or any species WGS?)? Is the limitation technical?

Only whole human germline applications are supported at this time TruPath, not somatic samples or non-human species.

Which sample types can be used for TruPath Genome?

TruPath workflow has been validated for blood and cell pellet samples. For additional samples, namely saliva, buccal swabs, and dried blood spots see TruPath Genome Performance with Samples of Varying Type and Quality tech note.

What blood anti-coagulant is preferred?

K2EDTA is the preferred anti-coagulant.

Can frozen blood be used?

High quality samples are fundamental for obtaining high quality DNA. In general, this comes from fresh blood samples stored up to three days at 4°C. Limited testing on frozen blood shows no major impact on proximity metrics. Nevertheless, refer to the extraction method manufacturer instructions for best practices.

Is TruPath suitable for cfDNA?

No, cfDNA size is typical around 150 bp. For TruPath, at a minimum, the sample should contain 50% of DNA fragments greater than 10 kb.

Is TruPath suitable for formalin-fixed paraffin-embedded (FFPE) tissues?

No, at a minimum, the sample should contain 50% of DNA fragments greater than 10 kb, which is usually not the case for FFPE samples. Additionally, FFPE samples can inhibit tagmentation on the flow cell so would also not be recommended even from purely a workflow perspective.

Is TruPath suitable for long amplicons (eg 1-10 kb)?

No, at a minimum, the sample should contain 50% of DNA fragments greater than 10 kb. Under this there is not a proximity data benefit to running these samples. Additionally, custom references are not supported, which would be required for amplicon or plasmid analysis.

What control sample is recommended to run if there is a problem with the assay to distinguish between sample/reagent issue? HG001 and HG002? Has Illumina tested Infinium DNA (PN 11191771)?

HG002 only is recommended as the control sample for troubleshooting, as it is possible to benchmark performance across a wider range of variant types.

Which kit or method should be used for DNA extraction?

High molecular weight DNA is optimal for TruPath workflow as there is a correlation between DNA quality and some of the improvements in variant calling performance. Extraction methods validated for blood and cells include Monarch HMW DNA (New England Biolabs). Additional methods tested for HMW DNA: Wizard HMW DNA (Promega) and MagAttract HMW DNA (Qiagen).

Standard molecular weight DNA methods can also be used, but will have reduced proximity information, including a lower Q25 proximity rate, which in turn translates to smaller template size, lower phasing block NG50, and lower percent of fully phased genes. Validated methods include QIAamp DNA blood mini (Qiagen). Additional methods tested for standard DNA, Mag-Bind Blood & Tissue DNA (Omega Bio-tek) and Chemagic DNA Universal kit H96 (Revvity).

Refer to the TruPath Genome Performance with Samples of Varying Type and Quality tech note for further information on DNA extraction from other sample types.

Which pipette tips can be used for handling high molecular weight DNA?

Wide bore tips are recommended for mixing HMW DNA prior to preparing the sample. If these are not available, pipette slowly using a P200 tip. Standard tips can be used for single pipetting steps (ie, drawing up the liquid and dispensing it only once, rather than mixing up and down which can cause shearing).

How should the DNA be stored?

DNA can be stored in the elution buffer provided with the respective extraction kit used, for a month at 4°C or at -20°C for long-term storage. Avoid more than 10 freeze/thaw cycles as this can degrade the DNA. Follow guidance per extraction kit recommendations per sample type and kit used.

What is the recommended input amount?

The recommended gDNA input is 350 ng. A range of 175 ng to 550 ng can be used with no major impact on coverage or other variant calling metrics. Lower input than this will reduce sample coverage and impact the variant calling performance benefits.

350 ng is recommended to ensure that even with a 50% inaccurate quantification then the input amount would not be within the recommended range.

How should the DNA input be QC'd?

DNA quantification and qualification methods are described in the DNA Input Recommendations of the Illumina TruPath Genome Product Documentation.

How to perform TapeStation region analysis?

Region analysis should be performed for accurate quantitation of fragment percentage larger than 10 and 60 kb. If using the region view, for the optimum sizing accuracy it is important to use a run ladder rather than an electronic ladder.

• On the TapeStation Analysis Software select Region

• Click on Region Settings

• To determine the area % >10 kb, set up the region from 10,000 bp to 500,000 bp. This allows for the max size possible. The fragment % > 10 kb can be taken from the software region table in % of Total (blue). For the sample shown below this is 92%.

• To determine the area % >60 kb, set up the region from 60,000 bp to 500,000 bp. This allows for the max size possible. The fragment % > 60 kb can be taken from the software region table in % of Total (orange). For this sample shown below this is 79%.

  

  

TapeStation region analysis for 10 and 60 kb fragment size.

Which input sources may give suboptimal results?

• Blood samples collected in sodium heparin.

• Certain cultured materials (amniocytes, Chorionic Villus Sampling (CVS)) combined with particular extraction methods (magnetic beads, salting out precipitation)

• Spin-column DNA purification methods yield lower quality DNA and therefore are not a preferred method; with the exception if required to removed contaminants.

• FFPE; due to size of fragments and poor tagmentation of damaged DNA/inhibition

• cfDNA; due to very small size of fragments

How to identify poor quality input from the analysis results?

Poor quality DNA will result in low Q25 proximity rate, proximity coverage, template lengths and phasing metrics. Extraction kit incompatibility and/or contaminant presents will most likely result in lower % PF, lower coverage, high duplicate percentage and potentially larger insert sizes.

For any feedback or questions regarding this article (Illumina Knowledge Article #10167), contact Illumina Technical Support [email protected].