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A) The effect of performing DASH directly on unwashed Klenow reactions in 50 µL (unwashed 50 µL rxn) as opposed to performing DASH after a beads cleanup in 20 µL (washed 20 µL rxn), on the detection (left) and read length (right) of spiked PDV (Phocine distemper virus), of non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), and CYB (cytochrome B), and of depleted ribosomal 18S and 28S RNA in metagenomic sequencing libraries of TURBO + DASH-depleted human serum samples. B) Increasing the Cas9 concentration lowers the loss of depletion observed when using direct DASH. C) Performing Klenow reactions in smaller volumes further reduces this loss of depletion

Journal: BMC Genomics

Article Title: Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform

doi: 10.1186/s12864-025-12268-4

Figure Lengend Snippet: A) The effect of performing DASH directly on unwashed Klenow reactions in 50 µL (unwashed 50 µL rxn) as opposed to performing DASH after a beads cleanup in 20 µL (washed 20 µL rxn), on the detection (left) and read length (right) of spiked PDV (Phocine distemper virus), of non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), and CYB (cytochrome B), and of depleted ribosomal 18S and 28S RNA in metagenomic sequencing libraries of TURBO + DASH-depleted human serum samples. B) Increasing the Cas9 concentration lowers the loss of depletion observed when using direct DASH. C) Performing Klenow reactions in smaller volumes further reduces this loss of depletion

Article Snippet: This ribonucleoprotein complex was formed by a 10 min preincubation at 37 °C of 1 μL Cas9 buffer, 2300 ng sgRNAs, and 3.4 μL Cas9 enzyme (M0386S, NEB) added up to 10 μL with water.

Techniques: Virus, Sequencing, Concentration Assay

The effect of omitting Cas9 inactivation by proteinase K (no protK) and subsequent heat inactivation at 95 °C (no 95 °C) on the detection of spiked PDV (Phocine distemper virus), non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), CYB (cytochrome B), ACTB (actin beta), and B2M (β2 microglobulin), and on the depletion of ribosomal RNAs 18S, 28S, and 16S in TURBO + DASH-depleted human serum samples. DASH was performed in a 40 µL volume directly on the second strand Klenow reactions

Journal: BMC Genomics

Article Title: Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform

doi: 10.1186/s12864-025-12268-4

Figure Lengend Snippet: The effect of omitting Cas9 inactivation by proteinase K (no protK) and subsequent heat inactivation at 95 °C (no 95 °C) on the detection of spiked PDV (Phocine distemper virus), non-depleted transcripts PGK1 (phosphoglycerate kinase 1), ND1/ND2/ND6 (NADH dehydrogenase subunit 1/2/6), COI (cytochrome c oxidase subunit I), CYB (cytochrome B), ACTB (actin beta), and B2M (β2 microglobulin), and on the depletion of ribosomal RNAs 18S, 28S, and 16S in TURBO + DASH-depleted human serum samples. DASH was performed in a 40 µL volume directly on the second strand Klenow reactions

Article Snippet: This ribonucleoprotein complex was formed by a 10 min preincubation at 37 °C of 1 μL Cas9 buffer, 2300 ng sgRNAs, and 3.4 μL Cas9 enzyme (M0386S, NEB) added up to 10 μL with water.

Techniques: Virus