Lysates were prepared as above. All reagents were sterilized, vacuum filtered, and autoclaved prior to use. DNA was purified using Purelink gDNA extraction kits (Thermo, #K182104A) as they improved yield in these low-biomass samples. The only modification to the Purelink protocol was elution of DNA into 10μL of sterile water three consecutive times, incubating the plate for 2 minutes at 37°C each time. DNA libraries were prepared using HackFlex (38 (link)) with the following modifications: (1) the tagmentation was scaled to start with 10μL of DNA; (2) the tagmentation-stop step was skipped; (3) we used standard Illumina barcodes for plates MG1-3 and UDI primers (20091660) for plates MG4-7 (Supplemental table 3 ); (4) we used KAPA HiFi master mix (Roche, 07958935001) and 19 cycles of PCR.
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Low-biomass DNA extraction and library prep
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Corresponding Organization : Broad Institute
Other organizations : Center for Systems Biology, Harvard University
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Variable analysis
independent variables
- DNA purification method (using Purelink gDNA extraction kits with modified elution protocol)
- DNA library preparation method (using HackFlex with modifications)
- DNA yield
- Sequencing results
- All reagents were sterilized, vacuum filtered, and autoclaved prior to use
- No positive or negative controls were explicitly mentioned in the provided information.
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