The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers
Abstract
:1. Introduction
2. Implications of Alternative Polyadenylation
3. Next-Generation Sequencing Based Techniques for Characterisation of APA
3.1. 3 focused RNA-seq Methods for APA Characterisation
3.2. Single-Cell Methods for mRNA 3 End Sequencing
4. Bioinformatic Methods for Detection of Poly(A) Sites
4.1. Databases for 3 UTR and APA Storage and Retrieval
4.2. Bioinformatic Methods for APA Detection and Quantification
4.2.1. APA Detection in RNA-seq Data Based on Prior APA Information
4.2.2. de novo APA Detection in RNA-seq Data
4.2.3. de novo APA Detection in 3 Focused Data
4.2.4. APA Detection in 3 Tag-Based Single-Cell RNA-seq Data
5. The Repertoire of Cancer Biomarkers
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Name | Key Points | Typical Input | Sequence Target |
---|---|---|---|
PAIso-seq [52] | PacBio based method to capture poly(A) site, length, splicing, expression, PacBio is costly for the read coverage obtained, Low coverage | 100 ng total RNA | Full length mRNA, Poly(A) tail included |
Oxford Nanopore- Direct RNA sequencing [53] | The Nanopore instrument is capable of full-length direct RNA seq, tail lengths can also be extracted. Low coverage | 500 ng poly(A)+ selected RNA | Full length mRNA, Poly(A) tail included |
TAIL-seq [54] | rRNA depletion and 3 adaptor ligation, asymmetric paired end sequencing to determine tail length | ∼100 g total RNA | Poly(A) tail length, Poly(A) site |
mTAIL-seq [55] | 3 oligo(dT) splinted ligation approach to TAIL-seq, reduced input RNA required. Paired-end sequencing. | 1–5 g total RNA | Poly(A) tail length, Poly(A) site |
PAT-seq [56] | Single end read approach, 3 tagging by oligo templated RNA end extension | 1 g total RNA | Poly(A) tail length, Poly(A) site |
PAL-seq [57] | Requires non-standard use of an Illumina instrument for tail length measurement by biotinylated dTTP incorporation. 3 end capture by splinted ligation | 1–50 g total RNA | Poly(A) tail length, Poly(A) site |
Poly(A) seq [58] | Poly(A)+ RNA is captured with oligo(dT) conjugated magnetic beads, then 3 adaptors ligated 300 bp single end read. Samples sequenced on the Illumina NextSeq 500, 2 colour sequencing instrument | 5.1 g total RNA | Poly(A) tail length, Poly(A) site |
TED-Seq [59] | 3 adaptor ligation to Poly(A)+ RNA. Tail length is inferred from the size of the templated sequence after precise library size selection | 100 ng poly(A)+ RNA | Poly(A) tail length, Poly(A) site |
3P-seq [4] | Poly(A) tail removed by RNase H. Sequenced from the 3 end to determine site usage, adaptor addition by ligation to avoid internal priming | 30 g total RNA | Poly(A) site |
2P-seq [60] | Poly(A) site detection by anchored oligo(dT) priming, sequencing from start of poly(A) tail in reverse | 15 g total RNA | Poly(A) site |
3-seq [30] | Poly(A) site detection by anchored oligo(dT) priming. Unique approach to fragmentation by rate limited nick translation of double stranded cDNA | 2 g DNase treated RNA | Poly(A) site |
3READS+ [37] | Poly(A) tail is trimmed by RNase H, 3 adapter ligation | 0.1–15 g total RNA | Poly(A) site |
3PC [61] | Anchored oligo(dT) primer to detect poly(A) site, 5 adaptor addition by circular ligation | 100 g total RNA | Poly(A) site |
3T-fill [62] | Anchored oligo(dT) primer to detect poly(A) site, sequenced from 3 end. 3T-fill reaction - dA homopolymer region at 3 end filled with dTTPs on Illumina cBot cluster station before sequencing | 0.5–10 g total RNA | Poly(A) site |
SAPAS [63] | Anchored oligo(dT) primer to detect poly(A) site, 5 adaptor addition by template switching | 10 g total RNA | Poly(A) site |
PAS-seq [5] | Anchored oligo(dT) primer to detect poly(A) site, template switching 5 adaptor addition | 0.5–1 g poly(A)+ selected RNA | Poly(A) site |
IVT-SAPAS [64] | in vitro transcription based amplification of cDNA for low input samples, poly(A) site detection by anchored oligo(dT) annealing | 200 ng total RNA | Poly(A) site |
PAPERCLIP [65] | RNA crosslinked, partially digested, and 3 ends immunoprecipitated via Poly(A) Binding protein, addresses internal priming issues, uses anchored oligo(dT) annealing for end detection | NA, starting material is tissue/cells | Poly(A) site |
MACE [66] | GenXPro commercial kit, barcodes transcripts with UMIs to deal with PCR duplication | 0.05 ng total RNA | Poly(A) site |
Quant-Seq [67] | Lexogen commercial kit, oligo(dT) annealing to detect 3 ends, random forward priming of 2nd strand cDNA adds 5 adaptor | 0.5–500 ng total RNA | Poly(A) site |
MAPS [68] | 3 end detection by anchored oligo(dT) priming, 5 adaptor addition by random forward priming of 2nd stand cDNA | 1 g total RNA | Poly(A) site |
TM3seq [69] | Fragmentation and 5 adaptor addition combined in a single step. 3 end detected via annealing of oligo(dT) primer | 200 ng total RNA | Poly(A) site |
PAC-seq [70] | Click-chemistry approach to fragmentation and 5 adaptor addition via reverse transcription termination by 3-azido-nucleotides. 3 end detected by oligo(dT) annealing | 0.125–4 g total RNA | Poly(A) site |
EnD-Seq [71] | Targeted sequencing approach to 3 end detection, 3 adaptor ligation to total RNA, gene specific multiplex PCR of cDNA | 1.5 g total RNA | Poly(A) site, non-Poly(A) 3 ends |
Name | Overview | Scale |
---|---|---|
CEL-seq [86] | 3 ends enriched by anchored oligo(dT) annealing including T7 promotor. cDNA amplified by in vitro transcription (IVT), amplified RNA fragmented and ligated to adaptor. | Manually isolated single cells |
CEL-seq2 [87,88] | Application of CEL-seq to high throughput sequencing, UMI’s added to reverse transcription oligo | Automated microfluidic sorting via Fluidigm C1 into wells |
MARS-seq 2.0 [89] | 3 end enrichment by anchored oligo(dT) annealing, included T7 promotor. cDNA amplified via IVT | 384-well plate, FACS sorting |
InDrop [80] | Application of CEL-seq to droplet-based sequencing for higher throughput | Droplet sequencing, inDrop system, 1CellBio |
Drop-seq [90] | 3 enrichment by oligo(dT) annealing RT, full length cDNA 5 labelled by template switching, oligo’s with common barcode bound to beads, and separated into droplets. library prepared by Illlumina Nextera XT DNA library prep kit | Droplet sequencing, custom instrument |
10X Chromium [85] | 3 enrichment by anchored oligo(dT) annealing, oligo’s with common barcode bound to beads, and separated into droplets; library preparation with commercial kit GemCode Single-Cell 3 Gel Bead and library kit (now Chromium 10X) | Droplet sequencing, 10X genomics instrument |
SCRB-seq [91] | 3 enrichment by anchored oligo(dT) primer, template switching reaction for full length cDNA, library prepared by Illlumina Nextera XT DNA library prep kit | 384-well plate, FACS sorting |
MAPS-seq [84] | 3 ends enriched by biotinylated oligo(dT) annealing, RNA transcripts pulled down and samples pooled together using magnetic beads before RT. Full length cDNA 5 adaptor added via template switching, library prepared by Illlumina Nextera XT DNA library prep kit | 96-well plate, FACS sorting |
BATSeq [92] | Method specifically developed to detect APA. 3 ends enriched by oligo(dT) annealing. 2nd strand cDNA IVT amplified | FACS sorting |
Database | Primary Data Collection | Organism | Last Updated | URL |
---|---|---|---|---|
UTRdb [93] | 5 and 3 UTR regions in EMBL/GenBank records | human, rodent, vertebrate, plant and fungi | 2010 | http://utrdb.ba.itb.cnr.it/ |
PACdb [95] | cDNA/ESTs | human, mouse, rat, dog, chicken, zebrafish, fugu, fruit fly, mosquito, nematode, Arabidopsis thaliana, rice and baker’s yeast | inaccessible | http://harlequin.jax.org/pacdb/ |
PolyA_DB [72,94,106] | aligned cDNA/ESTs | human, mouse, rat, chicken and zebrafish | 2018 | http://polya-db.org/v3/ |
GENCODE Poly (A) site track [100,101] | cDNA/ESTs | human | 2021 | https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeGencodeV19 |
APADB [97] | MACE-Seq | human, mouse and chicken | 2014 | http://tools.genxpro.net/apadb/ |
APASdb [96] | SAPAS | human (22 normal and cancer tissues), mouse, zebrafish and some lancelet samples | inaccessible | http://mosas.sysu.edu.cn/utr |
TC3A [99] | RNA-seq in TCGA | 32 human cancer types | inaccessible | http://tc3a.org/ |
APAatlas [27] | RNA-seq in GTEx project | >50 human normal tissue | 2020 | https://hanlab.uth.edu/apa/ |
PolyASite [98] | 3-Seq, 3READS, DRS, QuantSeq_REV, SAPAS, PAPERCLIP, PolyA-seq, PAS-Seq, A-seq, 3P-Seq, DRS, 2P-Seq, PAT-seq | human, mouse and worm | 2020 | https://polyasite.unibas.ch/ |
Cancer | Gene Markers | Signature APA | Physiological Effects | Molecular Role |
---|---|---|---|---|
Breast | PRELID1 | Shortening of 3 UTR | increased protein expression | mitochondrial ROS signalling [139] |
Breast | SNX3, YME1L1D, USP9X | Shortening of 3 UTR | increased protein levels in short isoform | EGF signalling [22] |
adult T-cell lymphoma, large B-cell lymphoma, stomach adenocarcinoma | PD-L1 gene (CD274) | Shortening of 3 UTR | PD-1/PD-L1-mediated immune escape in cancer development; structural variants (SVs) disrupt 3 regulatory region of PDL1 | T-cell modulator; PDCD1-mediated inhibitory pathway [136] |
Colorectal cancer | IGF2BP1/IMP-1 | Shortening of 3 UTR | increased protein levels; increased oncogenic transformation | Modulates pathogenesis [142] |
TNBC, lung, esophageal, bladder, leukemia, ovarian | N4BP2L2, WDHD1, ZER1, ADGRL2, PRSS12, NPL, SIK3, SYNGR1, SCL2A3, UBE2G2 | Shortening of 3 UTR | unfavourable prognosis | All are related to cancer development: cell cycle regulator and is involved in PI3K/Akt pathway; tumour antigen [138] |
TNBC | PPIC, ZCCHC14, RTN1, PRCK8, CLIC2, CXCL8, SMAD6 | Lengthening of 3 UTR | poor prognosis; response elements (MREs) in the lengthened 3 UTR leads to homologous gene repression and competing endogenous RNA (ceRNA) resulting in cancer progression; more miRNA binding sites | TGF-βpathway; autocrine NF-ƙB/IL-8 (CXCL8) pathway responsible for cell migration; aberrant pathways and cancer progression [138] |
TNBC (MB-231) | Caspase 6, DFFA (ICAD), DFFB (CAD), PARP1 | Lengthening of 3 UTR | escape of apoptosis | Caspase pathway [63] |
TNBC (MB-231) | cyclin D1, D2 | Shortening of 3 UTR | promote cell cycling | Mitotic cell cycle; APC [63] |
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Kandhari, N.; Kraupner-Taylor, C.A.; Harrison, P.F.; Powell, D.R.; Beilharz, T.H. The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers. Int. J. Mol. Sci. 2021, 22, 5322. https://doi.org/10.3390/ijms22105322
Kandhari N, Kraupner-Taylor CA, Harrison PF, Powell DR, Beilharz TH. The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers. International Journal of Molecular Sciences. 2021; 22(10):5322. https://doi.org/10.3390/ijms22105322
Chicago/Turabian StyleKandhari, Nitika, Calvin A. Kraupner-Taylor, Paul F. Harrison, David R. Powell, and Traude H. Beilharz. 2021. "The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers" International Journal of Molecular Sciences 22, no. 10: 5322. https://doi.org/10.3390/ijms22105322