RNA.md

Direct RNA and cDNA Sequencing of a human transcriptome on Oxford Nanopore MinION and GridION

Introduction

We have sequenced the CEPH1463 (NA12878/GM12878, Ceph/Utah pedigree) human genome reference standard on the Oxford Nanopore MinION using direct RNA sequencing kits (30 flowcells) and using the 1D ligation kit (SQK-LSK108) on R9.4 flowcells using R9.4 chemistry (FLO-MIN106). RNA from the GM12878 human cell line (Ceph/Utah pedigree) was extracted from the cultured cell line.

Contributors

• Winston Timp, Rachael Workman, Timothy Gilpatrick (Johns Hopkins)
• Jared Simpson, Phil Zuzarte, Paul Tang (OICR)
• Terry Snutch, John Tyson (UBC)
• Mark Akeson, Hugh Olsen, Benedict Paten, Angela Brooks, Miten Jain (UCSC)
• Nick Loman, Josh Quick, Andrew Beggs, Jaqueline Goes de Jesus (University of Birmingham)
• Matt Loose, Nadine Holmes, Matthew Carlile (University of Nottingham)

Acknowledgements

We are most grateful to Daniel Garalde, Daniel Jachimowicz, Andy Heron, Rosemary Dokos at Oxford Nanopore Technologies for technical and logistical assistance.

Analysis

Basecalls (Albacore 2.1)

Direct RNA (full dataset, 30 runs)

• Pass, 10302647 reads, mean 1030.24, N50 1334
• Fail, 2686736 reads, mean 430.96, N50 840

cDNA 1D (full dataset, 12 runs)

• Pass, 15152101 reads, mean 932.86, N50 1072
• Fail, 9129338 reads, mean 661.90, N50 841

Combined Albacore Summary

• Summary File (gzip)

Raw Signal Data

Direct RNA

All runs MinION unless noted.

• Bham Run1
• Bham Run2 (GridION)
• Bham Run3 (GridION)
• Bham Run4 (GridION)
• Bham Run5 (GridION)
• Hopkins Run1
• Hopkins Run2
• Hopkins Run3
• Hopkins Run4
• Hopkins Run5
• Notts_Run1
• Notts_Run2
• Notts_Run3
• Notts Run4
• Notts_Run5
• OICR Run1
• OICR Run2
• OICR Run3
• OICR Run4
• OICR Run5
• UBC Run1
• UBC Run2
• UBC Run3
• UBC Run4
• UBC_Run5
• UCSC Run1
• UCSC Run2
• UCSC Run3
• UCSC Run4
• UCSC Run5

Alignment Files

All alignments performed using minimap2.

Direct RNA

pass

• SIRVome BAM, SIRVome BAI
• cDNA Abinitio BAM, cDNA ab initio BAI
• Ensembl cDNA BAM, Ensembl cDNA BAI
• Ensembl CDS BAM, Ensembl CDS BAI
• Ensembl ncRNA BAM, Ensembl ncRNA BAI
• Genbank RNA BAM, Genbank RNA BAI
• hg38 BAM, hg38 BAI

fail

• SIRVome BAM, SIRVome BAI
• cDNA ab initio BAM, cDNA ab initio BAI
• Ensembl cDNA BAM, Ensembl cDNA BAI
• Ensembl CDS BAM, Ensembl CDS BAI
• Ensembl ncRNA BAM, Ensembl ncRNA BAI
• Genbank RNA BAM, Genbank RNA BAI
• hg38 BAM, hg38 BAI

Reference Files

• GRCh38 ab initio
• GRCh38 cDNA
• cDNA README
• GRCh38 CDS
• CDS README
• GRCh38 FASTA
• GRCh38 dict

GRC38 and BWA indices

• GRCh38 full analysis set plus decoy hla
• GRCh38 full analysis set plus decoy hla.alt
• GRCh38 full analysis set plus decoy hla.amb
• GRCh38 full analysis set plus decoy hla.ann
• GRCh38 full analysis set plus decoy hla.bwt
• GRCh38 full analysis set plus decoy hla.fai
• GRCh38 full analysis set plus decoy hla.pac
• GRCh38 full analysis set plus decoy hla.sai
• GRCh38 latest genomic.gff
• Homo_sapiens.GRCh38.90.abinitio.gtf
• Homo_sapiens.GRCh38.90.chr.gtf
• Homo_sapiens.GRCh38.90.chr_patch_hapl_scaff.gtf
• Homo_sapiens.GRCh38.90.gtf
• gtf_README

SIRVome

• SIRVome isoforms ERCCs 170612a.fasta
• SIRVome isoforms ERCCs Lot001485 C 170612a.gtf
• SIRVome isoforms ERCCs Lot001485 I 170612a.gtf
• SIRVome isoforms ERCCs Lot001485 O 170612a.gtf

cDNA

• NA12878 cDNA 1D pass BAM, NA12878-cDNA-1D.pass BAI
• NA12878 cDNA 1D fail BAM, NA12878-cDNA-1D.fail BAI

Run by run links

• 1D2 Bham Run1 all 1D only FASTQ
• 1D2 Bham Run2 all 1D only FASTQ
• 1D2 Hopkins Run1 all 1D only FASTQ
• 1D2 Hopkins Run2 all 1D only FASTQ
• 1D2 Notts Run1 all 1D only FASTQ
• 1D2 Notts Run2 all 1D only FASTQ
• 1D2 OICR Run1 all 1D only FASTQ
• 1D2 OICR Run2 all 1D only FASTQ
• 1D2 UBC Run1 all 1D only FASTQ
• 1D2 UBC Run2 all 1D only FASTQ
• 1D2 UCSC Run1 all 1D only FASTQ
• 1D2 UCSC Run2 all 1D only FASTQ
• 1D Bham Run1 fail FASTQ
• 1D Bham Run1 pass FASTQ
• 1D Bham Run2 fail FASTQ
• 1D Bham Run2 pass FASTQ
• 1D Hopkins Run1 fail FASTQ
• 1D Hopkins Run1 pass FASTQ
• 1D Hopkins Run2 fail FASTQ
• 1D Hopkins Run2 pass FASTQ
• 1D Notts Run1 fail FASTQ
• 1D Notts Run1 pass FASTQ
• 1D Notts Run2 fail FASTQ
• 1D Notts Run2 pass FASTQ
• 1D OICR Run1 fail FASTQ
• 1D OICR Run1 pass FASTQ
• 1D OICR Run2 fail FASTQ
• 1D OICR Run2 pass FASTQ
• 1D UBC Run1 fail FASTQ
• 1D UBC Run1 pass FASTQ
• 1D UBC Run2 fail FASTQ
• 1D UBC Run2 pass FASTQ
• 1D UCSC Run1 fail FASTQ
• 1D UCSC Run1 pass FASTQ
• 1D UCSC Run2 fail FASTQ
• 1D UCSC Run2 pass FASTQ
• DirectRNA Bham Run1 fail FASTQ
• DirectRNA Bham Run1 pass FASTQ
• DirectRNA Bham Run2 fail FASTQ
• DirectRNA Bham Run2 pass FASTQ
• DirectRNA Bham Run3 fail FASTQ
• DirectRNA Bham Run3 pass FASTQ
• DirectRNA Bham Run4 fail FASTQ
• DirectRNA Bham Run4 pass FASTQ
• DirectRNA Bham Run5 fail FASTQ
• DirectRNA Bham Run5 pass FASTQ
• DirectRNA Hopkins Run1 fail FASTQ
• DirectRNA Hopkins Run1 pass FASTQ
• DirectRNA Hopkins Run2 fail FASTQ
• DirectRNA Hopkins Run2 pass FASTQ
• DirectRNA Hopkins Run3 fail FASTQ
• DirectRNA Hopkins Run3 pass FASTQ
• DirectRNA Hopkins Run4 fail FASTQ
• DirectRNA Hopkins Run4 pass FASTQ
• DirectRNA Hopkins Run5 fail FASTQ
• DirectRNA Hopkins Run5 pass FASTQ
• DirectRNA Notts Run1 fail FASTQ
• DirectRNA Notts Run1 pass FASTQ
• DirectRNA Notts Run2 fail FASTQ
• DirectRNA Notts Run2 pass FASTQ
• DirectRNA Notts Run3 fail FASTQ
• DirectRNA Notts Run3 pass FASTQ
• DirectRNA Notts Run4 fail FASTQ
• DirectRNA Notts Run4 pass FASTQ
• DirectRNA Notts Run5 fail FASTQ
• DirectRNA Notts Run5 pass FASTQ
• DirectRNA OICR Run1 fail FASTQ
• DirectRNA OICR Run1 pass FASTQ
• DirectRNA OICR Run2 fail FASTQ
• DirectRNA OICR Run2 pass FASTQ
• DirectRNA OICR Run3 fail FASTQ
• DirectRNA OICR Run3 pass FASTQ
• DirectRNA OICR Run4 fail FASTQ
• DirectRNA OICR Run4 pass FASTQ
• DirectRNA OICR Run5 fail FASTQ
• DirectRNA OICR Run5 pass FASTQ
• DirectRNA UBC Run1 fail FASTQ
• DirectRNA UBC Run1 pass FASTQ
• DirectRNA UBC Run2 fail FASTQ
• DirectRNA UBC Run2 pass FASTQ
• DirectRNA UBC Run3 fail FASTQ
• DirectRNA UBC Run3 pass FASTQ
• DirectRNA UBC Run4 fail FASTQ
• DirectRNA UBC Run4 pass FASTQ
• DirectRNA UBC Run5 fail FASTQ
• DirectRNA UBC Run5 pass FASTQ
• DirectRNA UCSC Run1 fail FASTQ
• DirectRNA UCSC Run1 pass FASTQ
• DirectRNA UCSC Run2 fail FASTQ
• DirectRNA UCSC Run2 pass FASTQ
• DirectRNA UCSC Run3 fail FASTQ
• DirectRNA UCSC Run3 pass FASTQ
• DirectRNA UCSC Run4 fail FASTQ
• DirectRNA UCSC Run4 pass FASTQ
• DirectRNA UCSC Run5 fail FASTQ
• DirectRNA UCSC Run5 pass FASTQ

External Links

Heng Li has make a custom track for the UCSC genome browser from the direct RNA dataset. Thanks Heng! [1]

References

[1] Li, H Twitter link