This repository contains scripts used for the analysis performed in our manuscript
Improving RNA-seq protocols Felix Pförtner, Eva Briem, Wolfgang Enard, Daniel Richter
For the full prime-seq protocol please visit protocols.io.
We introduce an approach to optimize bulk RNA-seq protocols by systematically minimizing read loss across processing steps while keeping sensitivity and complexity uncompromised. We applied this "funnel strategy" to prime-seq, addressing critical stages of the protocol—including DNA digestion, reverse transcription, adapter ligation, and amplification—to achieve notable efficiency enhancements.
The optimized protocol increases final unique molecular identifiers (UMIs) by 60 % at equal sequencing costs or, conversely, reduces sequencing costs by 38 % at equal counts. This further improves one of the most cost-efficient bulk RNA-seq protocols available, creating prime-seq 2, and outlines strategies and mechanisms of potential relevance for other protocols amplifying complex nucleic acid samples for sequencing.
The main code creating the figures: Paper_Data_&_Plotting.Rmd
R version 4.4.1 (2024-06-14)
Platform: x86_64-pc-linux-gnu
Running under: Devuan GNU/Linux 4 (chimaera)
Matrix products: default
BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.13.so; LAPACK version 3.9.0
locale:
[1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
[7] LC_PAPER=en_US.UTF-8 LC_NAME=C LC_ADDRESS=C LC_TELEPHONE=C LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
time zone: Europe/Berlin
tzcode source: system (glibc)
attached base packages:
[1] stats graphics grDevices utils datasets methods base
loaded via a namespace (and not attached):
[1] vctrs_0.6.5 cli_3.6.5 rlang_1.1.6 purrr_1.0.4 car_3.1-2 generics_0.1.4 ggpubr_0.6.0 glue_1.8.0 backports_1.5.0
[10] rprojroot_2.0.4 scales_1.4.0 grid_4.4.1 abind_1.4-5 carData_3.0-5 tibble_3.2.1 rstatix_0.7.2 lifecycle_1.0.4 ggsignif_0.6.4
[19] compiler_4.4.1 dplyr_1.1.4 RColorBrewer_1.1-3 pkgconfig_2.0.3 tidyr_1.3.1 here_1.0.1 rstudioapi_0.16.0 farver_2.1.2 R6_2.6.1
[28] dichromat_2.0-0.1 tidyselect_1.2.1 pillar_1.10.2 magrittr_2.0.3 tools_4.4.1 gtable_0.3.6 broom_1.0.6 ggplot2_3.5.2