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Transcriptomics pilot data

The GEL Transcriptomics Pilot comprises RNA-sequencing of a subset of rare disease probands from the 100,000 Genomes Project who did not receive a genetic diagnosis through the Genomics England Interpretation Pipeline. We prioritised probands who were found to carry variants of unknown significance. The GEL Transcriptomics Pilot comprises RNA-sequencing of a subset (>5,000) of rare disease probands from the 100,000 Genomes Project who did not receive a genetic diagnosis through the Genomics England Interpretation Pipeline. We prioritised probands who were found to carry variants of unknown significance.

Protocol

Sequencing

  1. We derived total RNA from peripheral whole blood prepared using QIAGEN PAXgene Blood RNA Kit.
  2. We analysed isolated total RNA on an Agilent Tapestation 4200 system for RNA integrity number (RIN) and DV200 quality check.
  3. We constructed libraries following the Illumina TrueSeq Stranded mRNA Sample Prep, Ligation with Ribo-zero Plus kit protocol.
  4. We sequenced 95 samples per plate (one empty well) simultaneously on the NovaSeq 6000 using 2x100bp paired-end reads.

Data processing

We used the Illumina DRAGEN RNA Pipeline v3.8.4) for read alignment. We mapped reads to the human genome reference GRCh38 (alt-aware with HLAs) with annotation-assisted alignment, duplicate marking, gene expression quantification, and gene fusion detection enabled. We used GENCODE v32 GTF file for gene annotation. The Illumina DRAGEN aligner and gene expression quantifier are implementations of STAR and Salmon respectively.

Path to the reference genome in the RE:

/public_data_resources/reference/GRCh38DeAlt_HLA/GRCh38_full_analysis_set_plus_decoy_hla.fa

Path to the GTF file in the RE:

/public_data_resources/GENCODE/v32/GRCh38/gencode.v32.annotation.gtf

Data available in the Genomics England Research Environment

DRAGEN output

The data delivered by Illumina from running the DRAGEN RNA Pipeline is available in /gel_data_resources with individual deliveries subdivided by delivery dates (see example delivery below). You can generate lists of file paths of interest through the transcriptome_file_paths_and_types LabKey table by filtering for specific file type and participants.

Primary folder: /gel_data_resources/RNASeq_data/Rare_Disease/

For each sample the following output files are available:

/gel_data_resources/RNASeq_data/Rare_Disease/DELIVERY_DATE/DELIVERY_ID/
└── RNA_PLATEKEY
    ├── fastqs
    │   ├── RNA_PLATEKEY_SNN_L001_R1_001.fastq.gz
    │   ├── RNA_PLATEKEY_SNN_L001_R2_001.fastq.gz
    │   ├── ..
    │   ├── ..
    │   ├── RNA_PLATEKEY_SNN_L00N_R1_001.fastq.gz
    │   └── RNA_PLATEKEY_SNN_L00N_R2_001.fastq.gz
    ├── RNA_PLATEKEY.bam
    ├── RNA_PLATEKEY.bam.bai
    ├── RNA_PLATEKEY.fusion_candidates.final
    ├── RNA_PLATEKEY.quant.genes.sf
    ├── RNA_PLATEKEY.quant.sf
    ├── RNA_PLATEKEY.SJ.out.tab
    └── md5sum.txt

RNA-Seq QC output

We ran the sample-level RNA sequencing data through an internally developed pipeline, generating quality control metrics across the entire cohort. Evaluation included examining raw read quality, alignment quality, and whole genome DNA-RNA sample matching. Some sample-level output files from the below tools have been aggregated into simple tsv files which have been aggregated together (see below) to allow comparison across the full dataset.

The table below outlines the software employed by the pipeline for generating QC metrics and which output files fed into the aggregated files. FastQC, RNA-SeQC2, RSeQC were used to generate generic quality metrics, whereas Somalier was used to assess the relatedness between WGS and RNASeq data to ensure RNASeq samples matched the expected WGS data.

Tool Version Original output file types
FastQC 0.11.9 summary.txt
RNA-SeQC 2 2.4.2 *.bam.metrics.tsv
RSeQC 5.0.1 *.geneBodyCoverage.txt
Somalier 0.2.18 *.pairs.tsv
*.samples.tsv

Sample-level and aggregated QC files output by the pipeline can be accessed at:

/gel_data_resources/RNASeq_data/qc_results/Rare_Disease/<main_programme_data_release_version>

A subset of the QC metrics are available in the Labkey table accompanying this dataset, namely rnaseq_qc_metrics. This table can be used to screen samples based on their quality control metrics before conducting any subsequent analysis. Below is a breakdown of the metrics included in the table and the software used to generate them. These metrics were considered the most likely to provide an insight into the quality of a given sample, and thus most valuable during sample screening.

Column name Metric Description Software
participant_id Participant ID Genomics England participant identifier N/A
rnaseq_platekey RNA-Seq platekey Sample identifier for RNASeq data N/A
wgs_platekey WGS platekey Sample identifier for WGS data N/A
rna_folder_path RNA (data) folder path Path to the folder containing RNASeq data for a given sample N/A
rnaseqc_total_reads Total Reads Total input alignments RNA-SeQC 2
rnaseqc_mapping_rate Mapping rate Proportion of Mapped Reads (Unique Mapping, Vendor QC Passed Reads that were mapped) to total reads RNA-SeQC 2
rnaseqc_duplicate_rate_of_mapped Duplicate rate of mapped Proportion of Mapped Duplicate Reads to Mapped Reads RNA-SeQC 2
rnaseqc_high_quality_rate High quality rate Mapped Reads that passed the following criteria: aligned as proper pairs, mismatches (NM tag) at or below threshold (--base-mismatch 6), passed mapping quality (MAPQ) threshold (--mapping-quality 60) RNA-SeQC 2
rnaseqc_exonic_rate Exonic rate Proportion of Exonic Reads (mapped Reads for which all aligned segments unambiguously aligned to exons of the same gene) among Mapped Reads RNA-SeQC 2
rnaseqc_intronic_rate Intronic rate Proportion of Intronic Reads (mapped Reads for which all aligned segments unambiguously aligned to the same gene, without intersecting any of its exons) among Mapped Reads RNA-SeQC 2
rnaseqc_intergenic_rate Intergenic rate Proportion of Intergenic Reads among Mapped Reads RNA-SeQC 2
rnaseqc_rrna_rate rRna rate Proportion of rRNA Reads among Mapped Reads RNA-SeQC 2
rnaseqc_fragment_length_median Fragment length median Median insert size RNA-SeQC 2
rnaseqc_median_3_prime_bias Median 3' bias Median 3’ bias in read coverage RNA-SeQC 2
rnaseqc_median_exon_cv Median exon CV Median coefficient of variation of exon coverage, computed excluding the first and last 500bp of genes, using High Quality Reads mapping fully and exclusively to the gene RNA-SeQC 2
rnaseqc_genes_detected Genes detected Number of genes with at least five (default) High Quality Exonic Reads (--detection-threshold 5) RNA-SeQC 2
rseqc_gene_body_coverage_skewness Gene body coverage skewness Fishers coefficient of skewness of the gene body coverage calculated by RSeQC RSeQC (further calculated with "moments")
somalier_relatedness Relatedness A measure of the degree the similarity of the genotypes at polymorphic loci between matched transcriptomic and WGS DNA samples. Somalier
somalier_x_depth_mean X depth mean Mean depth of sites on X chromosome Somalier
somalier_y_depth_mean Y depth mean Mean depth of sites on Y chromosome Somalier
somalier_x_n X n (sites) Number of sites available on X chromosome in RNAseq sample. This is based on a maximum number of chrX sites present in /public_data_resources/somalier/v0.2.18/sites.*.vcf.gz Somalier
somalier_y_n Y n (sites) Number of sites available on Y chromosome in RNAseq sample. This is based on a maximum number of chrY sites present in /public_data_resources/somalier/v0.2.18/sites.*.vcf.gz Somalier
somalier_x_hom_ref X hom ref Number of chrX homozygote reference sites found in RNAseq sample. Somalier
somalier_x_hom_alt X hom alt Number of chrX homozygote alternate sites found in RNAseq sample. Somalier
somalier_x_het X het Number of chrX heterozygote sites found in RNAseq sample. Somalier
illumina_rin RIN RNA Integrity Number as measured by an Agilent Tapestation 4200. Values were not used to remove low quality samples before sequencing. All samples were sequenced regardless of their quality in order to define RIN/DV200 thresholds to be used in future RNA-seq projects. Illumina Provided
illumina_rin DV200 DV200 (percentage of fragments >200 nt) as measured by an Agilent Tapestation 4200. Values were not used to remove low quality samples before sequencing. All samples were sequenced regardless of their quality in order to define RIN/DV200 thresholds to be used in future RNA-seq projects. Illumina Provided

Quality Assessment Summary

From a subset of the QC metrics we provide a summarised overview below. Two samples were observed with extremely high number of reads, and have been excluded from the figures below.

Summary results of the RNASeQ QC pipeline with alignment metrics. Also here, the two samples with extremely high number of reads have been excluded from this table.

Metric Min Q1 Median Mean Q3 Max
rnaseqc_total_reads 112,939,429 217,359,043 248,518,554 255,393,099 285,917,493 956,819,802
rnaseqc_duplicate_rate_of_mapped 0.112 0.376 0.489 0.504 0.615 0.979
rnaseqc_exonic_rate 0.172 0.27 0.289 0.293 0.311 0.694
rnaseqc_fragment_length_median 145 176 186 186.878 196 264
rnaseqc_genes_detected 10,731 24,943 25,999 25,685.400 26,818.750 41,243
rnaseqc_high_quality_rate 0.777 0.924 0.928 0.925 0.93 0.94
rnaseqc_intergenic_rate 0.023 0.049 0.051 0.051 0.053 0.256
rnaseqc_intronic_rate 0.25 0.597 0.62 0.616 0.638 0.727
rnaseqc_mapping_rate 0.64 0.953 0.966 0.96 0.975 0.995
rnaseqc_median_3_prime_bias 0.336 0.47 0.491 0.489 0.507 0.643
rnaseqc_median_exon_cv 0.27 0.328 0.348 0.355 0.373 0.751
rnaseqc_rrna_rate 0 0 0 0 0 0.024
rseqc_gene_body_coverage_skewness -2.141 -1.158 -1.083 -1.091 -1.014 -0.494

Finally, FastQC results are summarised in the table below showing the proportion of flagged samples per category across ten fastQC modules.

Category FAIL WARN PASS
Adapter Content 0.5 1.2 98.3
Overrepresented sequences 1.3 7.6 91
Per base N content NA 0.1 99.9
Per base sequence content 9.3 90.7 NA
Per base sequence quality 93.5 NA 6.5
Per sequence GC content 53.3 44.4 2.3
Per sequence quality scores NA NA 100
Per tile sequence quality 0.6 0.4 98.9
Sequence Duplication Levels 99.5 0.5 NA
Sequence Length Distribution NA 100 NA

Help and support

Please reach out via the Genomics England Service Desk for any issues related to the RNA-Seq datasets and tables, including "RNASeq" in the title/description of your inquiry.

f7193d9 (RNAseq page)