Figure 4
From: High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing
Unique molecular identifiers (UMIs) collapse duplicate reads and reveal linear relationship at low PCR cycles between total and collapsed counts, but drop out at high PCR cycles. (A-C) Effect of increasing length of UMI on number of miRNA counts following collapsing of miRNA + UMI, compared to total “raw” count. Number to left of + sign represents Ns on the 5′ adapter while numbers to right represent 3′ adapter. Insert represent collapse on miRNAs alone (i.e. without and UMI). Raw represents uncollapsed read count. Analysis shown for all miRNAs (A), a highly expressed miRNA (miR-21, B) and intermediately expressed miRNA (miR-96, C). (D) Correlation plot of log10 counts per million for each miRNA comparing collapsed versus uncollapsed (total) reads for a library amplified for 14 cycles. All 12 random nucleotides were used for collapsing. (E) Same as D, but amplified for 24 cycles showing reduction in correlation for low expressed miRNAs. (F) Same as D, but comparing only collapsed reads between library amplified for 14 versus 24 cycles, showing much poorer correlation for low to intermediate expressed miRNAs. (G-I) Direct comparison of read counts for each miRNA from libraries differing in the number of PCR amplification cycles. miRNAs are ordered from high to low expression in 14 cycle library. (G) Uncollapsed (total) counts per million. (H) Collapsed counts per million. (I) Percent unique reads (i.e. collapsed counts/total counts *100). Note noise created by high PCR cycle number on the lowly to intermediately expressed miRNAs. All libraries were made from one cellular input RNA.
