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| Status |
Public on Feb 08, 2019 |
| Title |
ONT_683_SupCol |
| Sample type |
SRA |
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| Source name |
Brain tissue
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| Organism |
Homo sapiens |
| Characteristics |
brain region: Superior colliculus
donor: A
age: 64
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the QIAGEN miRNeasy kit.
RNA CaptureSeq was performed as per the protocol from Mercer, T.R. et al Nature Protocols 9, 989-1009 (2014), with several modifications for the ONT platform. 1 µg of each of 4 human brain samples was spiked with RNA sequins (Mix A) at 2% fractional abundance. Following reverse transcription, PCA adapters from the ONT ligation sequencing kit 1D (SQK-LSK108) were ligated onto the end-prepped samples. The captured samples were barcoded using ONT 1D native barcoding genomic DNA kit (EXP-NBD103), and library prep was performed using the 1D genomic DNA by ligation kit and protocol for the PromethION sequencer (SQK-LSK109).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
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| Data processing |
ONT reads were base-called using Guppy base-calling software (v1.8.5).
Demultiplexing base-called reads was performed using Porechop (v0.2.3) with the enforced barcode detection parameter.
Reads aligned to hg38 (plus in silico chromosome) using minimap2 (v2.14-r883).
To generate a hybrid transcriptome that leverages both long- and short-read data, we used the FLAIR tool (v1.2). First, we used FLAIR ‘correct’ by inputting spliced ONT read alignments (from minimap2) and correcting misaligned ONT splice junctions using genome annotations (GENCODE v29; with the –f option) and our accompanying Illumina splice junctions (with the –j option). We used the default window size of 10 bp for correcting splice sites. We then collapsed corrected reads into a non-redundant transcriptome using FLAIR ‘collapse’ with default parameters. We then undertook a number of steps in order to produce a high-confidence set of multi-exonic transcripts overlapping GWAS haplotype blocks associated with neuropsychiatric functions.
We quantified transcript expression using Salmon (v0.11.3), providing the chrIS annotation and our hybrid transcriptome as a reference. For ONT samples, we used the following non-default parameters: --fldMean 1000 --fldSD 100 --libtype U.
Genome_build: hg38
Supplementary_files_format_and_content: BED file contains chromosome coordinates of all transcripts in our filtered hybrid transcriptome. The four '*_quant.sf' files provide Salmon expression measurements (TPM) for hybrid transcriptome and RNA sequins.
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| Submission date |
Feb 07, 2019 |
| Last update date |
Feb 12, 2019 |
| Contact name |
Simon Andrew Hardwick |
| E-mail(s) |
[email protected]
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| Phone |
+61401264672
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| Organization name |
Garvan Institute of Medical Research
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| Lab |
Transcriptomic Research
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| Street address |
384 Victoria Street
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| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE118158 |
Targeted, long-read RNA sequencing of non-coding genomic regions associated with neuropsychiatric functions. |
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| Relations |
| BioSample |
SAMN10887167 |
| SRA |
SRX5350538 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3595457_ONT_683_SupCol_quant.sf.txt.gz |
304.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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