Supplementary Data - Nanopore cDNA With and Without mtDNA

Update (v1.5.8-SUPcaller): added scripts and data files for mt-Nd1 comparisons for different basecaller versions:

• LAST_gci_*.csv - gap-compressed identity statistics
• compStats_*.csv - called base-level statistics
• process.txt - script example file demonstrating how to generate statistics
• smealgol.r - R script to generate gap-compressed identities from LAST output
• est_vs_actual.png - density plots comparing gap-compressed identity with mean estimated accuracy from the caller
• GCI_albacore_guppy.png - violin plots comparing gap-compressed identity with different callers
• BaseQ_vs_GCI.png - scatter plots comparing gap-compressed identity with mean estimated accuracy from the caller

Update (v1.5.7-Nd1Reads): added mt-Nd1 read subset from CGDec2017 run:

• 4T1-WT_reads_CGDec2017_singlePlusMulti.tar

Update (v1.5.6-countAnalysis): added / updated a script to convert transcript mappings to a count table:

• count_analysis.r

Update (v1.5.5-Int): added intermediate (post-demultiplexing) demonstration dataset:

• SampleDESet_60k_demultiplexed.tar.gz

Also updated count tables and DESeq2 results:

• raw_counts_2019-Nov-29.csv
• DE_orig_VST_GRCm38_CG_GL261_2019-Nov-29.csv.gz
• DE_shrunk_VST_GRCm38_CG_GL261_2019-Nov-29.csv.gz

Update (v1.5.4-MT): updated demonstration dataset (now 40Mb) to include mitochondrial transcripts, and exclude non-target sequences from chimeric reads:

• reads_SampleDESet_sampled_60k.fastq.gz
• Mm_subset.GRCm38.ensembl_v98.fa

Update (v1.5.3-Ensembl): added/updated Ensembl reference transcripts, including non-coding RNA:

• ensembl_mm10_geneFeatureLocations.txt.gz - genome annotation
• Mus_musculus.GRCm38.ensembl_v98.fa.gz - transcript sequences

Update (v1.5.2-Demo): added 60Mb demonstration dataset for teaching / learning purposes (reads_SampleDESet_sampled_60k.fastq.gz). This dataset can be used for testing demultiplexing, or carrying out differential expression analysis on nanopore reads. There are 15k reads sampled from each run (subset on ~300 genes), plus 7.5k chimeric reads from the Dec17 run, plus 7.5k reads sampled from from CG009/BC08. The gene subset includes ~100 genes differentially-expressed between 4T1WT and 4T1ρ⁰ cell lines, plus an additional ~200 genes with minimal differential expression observed between any 4T1 cell lines (i.e. a housekeeping gene set):

• BC01, BC02, BC04, BC06 - 4T1 cell line reads from three sequencing runs (005, 005, 004, Dec17)
• BC03, BC05, BC07 - 4T1ρ⁰ (without mtDNA) cell line reads from three sequencing runs (005, 004, Dec17)
• BC08 - 4T1ρ⁰SC (from subcutaneous tumour) cell line reads from one sequencing run (009)

[note: v1.5.1-Demo has the wrong sequence ID for BC08 reads]

Update (v1.5.0-Called): added full 4T1 call set (called using guppy high-accuracy v3.2.2), gene transcript count tables, and differential expression results

Update (v1.4.1-Chimeric): added more example called chimeric reads of various [unclassified] categories

Update (v1.4.0-Chimeric): added example chimeric reads, one formed [presumably] during sample prep ["conjugation"], another formed in-silico ["double read"]

Update (v1.3.0-DESeq2): added count table and metadata file for DESeq2 analysis

Update (v1.2.1-10k): added 10k of FASTQ reads from a more recent run (part of the same differential expression project).

Supplementary data to go with David Eccles' poster about nanopore cDNA sequencing with and without mitochondrial DNA.

Reference mouse mtDNA and mtDNA reads from 4T1 cells (from MinION cDNA sequencing, and from SRA run SRR6747859).

• fwd_4T1_BC06.correctedReads.uniqueOnly.fasta.gz -- Canu-corrected cDNA reads from Wildtype 4T1 strain, filtered as transcript-forward orientation
• rev_4T1_BC06.correctedReads.uniqueOnly.fasta.gz -- Canu-corrected cDNA reads from Wildtype 4T1 strain, filtered as transcript-reverse orientation
• fwd_4T1_BC07.correctedReads.uniqueOnly.fasta.gz -- Canu-corrected cDNA reads from 4T1ρ0 strain (no mitochondrial DNA), filtered as transcript-forward orientation
• rev_4T1_BC07.correctedReads.uniqueOnly.fasta.gz -- Canu-corrected cDNA reads from 4T1ρ0 strain (no mitochondrial DNA), filtered as transcript-reverse orientation

Other intermediate data files are also included, see `4way_mapping_process.txt` for more details.

The mpileup differential coverage script can be found here.