> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-faq-list/000010167.md).
# Input recommendations FAQ for the TruPath Genome assay
**Which species are supported for TruPath sequencing?**
Only whole human germline applications are supported at this time with TruPath Genome, not somatic samples or non-human species.
**Which sample types can be used for TruPath Genome?**
The TruPath workflow has been validated for blood and cell pellet samples. For additional samples, namely saliva, buccal swabs, and dried blood spots see the [TruPath Genome Performance with Samples of Varying Type and Quality tech note](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trupath-genome-sample-info-tech-note-m-gl-03932/trupath-genome-sample-info-tech-note-m-gl-03932.pdf).
**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 may inhibit tagmentation on the flow cell, making them unsuitable from a workflow standpoint as well.
**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 no 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?**
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 the TruPath Genome 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 include 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 include 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](https://www.illumina.com/content/dam/illumina/gcs/assembled-assets/marketing-literature/trupath-genome-sample-info-tech-note-m-gl-03932/trupath-genome-sample-info-tech-note-m-gl-03932.pdf) for further information on DNA extraction from other sample types.
**Which pipette tips can be used for handling high molecular weight (HMW) 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 one 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 the extraction kit guidelines based on the sample type and the 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, the input amount would still 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](https://support.illumina.com/downloads/trupath-genome-product-documentation-200065852.html).
**How to perform TapeStation region analysis?**
Region analysis should be performed for accurate quantitation of fragment percentage larger than 10 kb 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.
* Select 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%.
**Figure 1**. TapeStation region analysis for 10 and 60 kb fragment sizes.
**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 typically produce lower-quality DNA and are therefore not preferred, except when necessary to remove 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.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #10167), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000010167%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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