Experiment / E18LA7OSF5' UTR / Translation Efficiency MPRA (MPTA)

Integrated Toxoplasma 5′ UTR MPRA

5′ untranslated regions tune Toxoplasma translation

A 50,000-member synthetic 5′ UTR library derived from 12 endogenous Toxoplasma UTRs was cloned upstream of mNeonGreen in an RH reporter, targeted to a neutral chromosome VI locus, and sorted by mKate-positive/mNeonGreen intensity into four bins. Two biological replicates were sequenced and author QC yielded MPRA scores for 30,235 unique sequences.

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Organism: NCBITaxon:5811
Taxonomy ID: NCBITaxon:5811
Biosample: UNMAPPED:Toxoplasma_gondii_RH_dKu80_dHXGPRT
Reference genome: Not reported / not applicable
Design focus: Synthetic / Motif-focused
Region of interest: Not reported / not applicable

Perturbation & assay details

Basal / Untreated

Linearized pMLP160 reporter was co-transfected with a Cas9 plasmid into RH parasites for homologous recombination at a neutral chromosome VI locus. The reporter contained the TUB1 promoter plus the first 50 nt of the TUB1 5′ UTR, the variable library sequence, an mNG-CDPK3 3′ UTR, and a constitutive mKate2-T2A-DHFR selection/gating cassette; parasites were selected with 1.5 µM pyrimethamine, sorted into mNG-negative and bottom/middle/top 15% positive bins, and scored from targeted DNA sequencing. Because high-quality RNA libraries were difficult to recover from sorted populations, RNA/DNA stability QC used bulk populations only.

Processed data

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Filters apply to this table only. The CSV download contains the complete processed table; filtered rows are available through the API.

Column dictionary · 25 definitions

element_id: Unique synthetic 5′ UTR library sequence identifier, such as seq_7.
sequence: DNA sequence of the tested 5′ UTR library element as represented in the GEO/MPRA score table; the reporter also contained a constant TUB1 5′ UTR context described in the paper.
sequence_length: Length of the submitted UTR sequence in nucleotides.
uaug_count: Number of ATG (uAUG) triplets in the sequence column, i.e. the sequenced library insert.
gc_fraction: Fraction of sequence bases that are G or C.
mpra_score: Author-calculated MPRA score: the mean of a fitted normal distribution over the four FACS reporter-intensity bins, with mKate-positive/mNG-negative = 0, bottom 15% mNG-positive = 1, middle 15% = 2, and top 15% = 3.
bulk_dna_rep1_cpm: Author-provided normalized bulk parasite DNA abundance for biological replicate 1, in counts per million.
bulk_dna_rep2_cpm: Author-provided normalized bulk parasite DNA abundance for biological replicate 2, in counts per million.
bulk_dna_mean_cpm: Arithmetic mean of the two author-provided bulk DNA counts-per-million values.
bulk_dna_rep1_raw_count: Count for this sequence in the GEO counted-read archive for bulk parasite DNA replicate 1, before counts-per-million normalization.
bulk_dna_rep2_raw_count: Count for this sequence in the GEO counted-read archive for bulk parasite DNA replicate 2, before counts-per-million normalization.
mng_negative_rep1_cpm: Author-provided DNA abundance in the mKate-positive/mNG-negative FACS bin for replicate 1, normalized to matched bulk DNA counts per million.
mng_negative_rep2_cpm: Author-provided DNA abundance in the mKate-positive/mNG-negative FACS bin for replicate 2, normalized to matched bulk DNA counts per million.
mng_negative_mean_cpm: Arithmetic mean of the two normalized mKate-positive/mNG-negative bin values.
mng_bottom_rep1_cpm: Author-provided DNA abundance in the bottom 15% mNG-positive FACS bin for replicate 1, normalized to matched bulk DNA counts per million.
mng_bottom_rep2_cpm: Author-provided DNA abundance in the bottom 15% mNG-positive FACS bin for replicate 2, normalized to matched bulk DNA counts per million.
mng_bottom_mean_cpm: Arithmetic mean of the two normalized bottom-bin values.
mng_middle_rep1_cpm: Author-provided DNA abundance in the middle 15% mNG-positive FACS bin for replicate 1, normalized to matched bulk DNA counts per million.
mng_middle_rep2_cpm: Author-provided DNA abundance in the middle 15% mNG-positive FACS bin for replicate 2, normalized to matched bulk DNA counts per million.
mng_middle_mean_cpm: Arithmetic mean of the two normalized middle-bin values.
mng_top_rep1_cpm: Author-provided DNA abundance in the top 15% mNG-positive FACS bin for replicate 1, normalized to matched bulk DNA counts per million.
mng_top_rep2_cpm: Author-provided DNA abundance in the top 15% mNG-positive FACS bin for replicate 2, normalized to matched bulk DNA counts per million.
mng_top_mean_cpm: Arithmetic mean of the two normalized top-bin values.
bulk_rna_rep1_umi_count: Bulk reporter RNA UMI count for replicate 1 from the GEO counted-read archive; included as a QC/context measure and not as a sorted-bin RNA measurement.
bulk_rna_rep2_umi_count: Bulk reporter RNA UMI count for replicate 2 from the GEO counted-read archive; included as a QC/context measure and not as a sorted-bin RNA measurement.

Quality control

Reads were trimmed with cutadapt, merged with fastq-join, and unique 170–190-nt UTR sequences were retained only if detected in both unsorted bulk-DNA replicates and the input plasmid library. DESeq2 compared de-duplicated bulk RNA with bulk DNA; UTRs with adjusted p < 0.05 were excluded. Remaining UTRs required at least 25 reads in each bulk DNA replicate and at least 25 total reads across the four sorted bins in each replicate; sample counts were converted to counts per million and bin values were normalized to matched bulk counts per million. The processed table contains all 30,235 author-passing sequences; package validation confirmed A/C/G/T-only sequence, length 170–190 nt, unique IDs and sequences, finite 0–3 MPRA scores, and all raw-count thresholds.

Curation notes

This is a synthetic sequence library, not a GWAS or allele-pair experiment. The GEO score table and Supplementary Data 5 contain the same 30,235 sequence-level MPRA results. The uaug_count field is computed only from the sequence column; the reporter also had a constant TUB1 5′ UTR context described in the paper, which is not separately appended or modeled in this table. Bulk RNA UMI counts are supplied as context from the counted-read archive; MPRA bin values are author-provided counts per million normalized to corresponding bulk DNA, and mpra_score is the fitted-bin-distribution mean. No discrete genomic region or named assembly was specified for the synthetic library, so reference_genome and region_of_interest are null.

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