Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Authors: Haiqi Xu^1,2,*, Linzhen Kong^1,2,*, Jingfei Cheng^1,2, Khatoun Al Moussawi¹, Xiufei Chen^1,2, Aleema Iqbal^1,2, Peter A. C. Wing³, James M. Harris⁴, Senko Tsukuda⁴, Azman Embarc-Buh⁵, Guifeng Wei⁶, Alfredo Castello⁵, Skirmantas Kriaucionis¹, Jane A. McKeating⁴, Xin Lu¹, and Chun-Xiao Song^1,2,†

Introduction

These scripts are for BACS data analysis. The following steps are included:

1. Data preprocessing
2. Alignment and filtering
3. Mutation counts and site calling

Spike in

NNUNN1(fully modified NNΨNN) GCTTCAAGTTGANNTNNCATCGCAAGTGCA

NNUNN2(unmodified NNUNN) ATGTCTCGACGTNNTNNGTTACAGTACCGT

Reference

Hg38 for human samples; For RNA viruses, the following reference genomes were used:

Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1 (NC_045512.2),

Recombinant Hepatitis C virus J6(5’UTR-NS2)/JFH1 (JF343782.1),

Zika virus isolate ZIKV/H.sapiens/Brazil/Natal/2015 (NC_035889.1),

Hepatitis Delta Virus sequence from the pSVL(D3) plasmid99 (Addgene plasmid #29335) (https://www.addgene.org/29335/), and Sindbis virus (NC_001547.1).

For EBV samples, reads were aligned to Epstein-Barr virus (EBV) genome, strain B95-8 (V01555.2)

1. Data preprocessing, alignments, filtering, and mutation counts

For Ribo-depletion libraries: bacs_smallrna.smk, bacs_smallrna_callsite.smk

For polyA libraries: bacs_alignment1.smk bacs_mRNA_part2.smk and bacs_mRNA_site.smk

2. Call high-confidence pseudouridine sites for small RNA and mRNA

calling_mRNA_ivt1.r and site_calling.r

3. Results for spike-ins( Fig. 1 c,d and Supplementary Fig. 1 c,d)

figure_spikein.r

4. Results for rRNA and snRNA (Fig2 b-I, Supplementary Fig. 2,3,4)

figure2.r

5. Results for snoRNA (Supplementary Fig. 5, 6)

figure_snoRNA.r

6. Results for tRNA (Fig3, Supplementary Fig. 7,8,12)

figure_tRNA.r

7. Results for mRNA (Fig4, Supplementary Fig. 9,10)

figure_mRNA.r inosine_plot.r

8. Results for comparision between different methods (Supplementary Fig. 11)

comparision_method.r

9. Results for KO (Fig 5,Supplementary Fig. 13)

knockout.r

10. Results for virus samples (Fig 6,Supplementary Fig. 14,15 )

virus_final.r