scholarly journals Full-length poly(A) and mRNA sequencing (FLAM-seq)

2019 ◽  
Author(s):  
Ivano Legnini ◽  
Jonathan Alles ◽  
Nikos Karaiskos ◽  
Salah Ayoub ◽  
Claudia Quedenau ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tail Length ◽  
Full Length ◽  
Cdna Libraries ◽  
Simple Method ◽  
Mrna Sequencing ◽  
New Strategy ◽  
Long Read ◽  
Mrna Isoform ◽  
Cdna Preparation

Abstract We developed FLAM-seq, a fast and simple method for generating cDNA libraries of full-length mRNAs, including the poly(A) tail. By combining a new strategy for cDNA preparation with PacBio long-read sequencing, FLAM-seq enables to generate hundreds of thousands of reads per sample in an easy and short procedure, with starting material ranging from 500 ng to 10 μg of total RNA. Besides the quantification of gene expression and the information about the mRNA isoform, the data generated with FLAM-seq allow accurate measurement of poly(A) tail length for thousands of genes in the sequenced samples. Here we describe the protocol step by step from the RNA preparation to the first steps of data analysis.

scholarly journals Full-Length Enrich c-DNA Libraries-Clear Cell-Renal Cell Carcinoma

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Sai-Wen Tang ◽  
Jung-Yaw Lin
Keyword(s):  
Gene Expression ◽  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Molecular Mechanisms ◽  
Clear Cell ◽  
Expression Profiles ◽  
Full Length ◽  
Cdna Libraries ◽  
Cell Renal Cell Carcinoma

Clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, is characterized by high metastasis potential and strong resistance to traditional therapies, resulting in a poor five-year survival rate of patients. Several therapies targeted to VEGF pathway for advanced RCC have been developed, however, it still needs to discover new therapeutic targets for treating RCC. Genome-wide gene expression analyses have been broadly used to identify unknown molecular mechanisms of cancer progression. Recently, we applied the oligo-capping method to construct the full-length cDNA libraries of ccRCC and adjacent normal kidney, and analyzed the gene expression profiles by high-throughput sequencing. This paper presents a review for recent findings on therapeutic potential of MYC pathway and nicotinamide N-methyltransferase for the treatment of RCC.

scholarly journals FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control

2019 ◽  
Vol 16 (9) ◽  
pp. 879-886 ◽  
Author(s):  
Ivano Legnini ◽  
Jonathan Alles ◽  
Nikos Karaiskos ◽  
Salah Ayoub ◽  
Nikolaus Rajewsky
Keyword(s):  
Tail Length ◽  
Full Length ◽  
Mrna Sequencing

scholarly journals Full-length mRNA sequencing reveals principles of poly(A) tail length control

2019 ◽  
Author(s):  
Ivano Legnini ◽  
Jonathan Alles ◽  
Nikos Karaiskos ◽  
Salah Ayoub ◽  
Nikolaus Rajewsky
Keyword(s):  
Single Molecule ◽  
Alternative Polyadenylation ◽  
Tail Length ◽  
Full Length ◽  
Alternative Promoters ◽  
High Quality ◽  
Simple Method ◽  
Single Molecule Sequencing ◽  
C Elegans ◽  
Full Length Sequence

SummaryAlthough mRNAs are key molecules for understanding life, there exists no method to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although poly(A) tails can be modified in functionally important ways, there also exists no method to accurately sequence them. Here, we present FLAM-seq, a rapid and simple method for high-quality sequencing of entire mRNAs. We report a cDNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids, and C. elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that (a) 3’ UTR length is correlated with poly(A) tail length, (b) alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, (c) tails contain a significant number of cytosines. Thus, we provide a widely useful method and fundamental insights into poly(A) tail regulation.

scholarly journals HIT-scISOseq: High-throughput and High-accuracy Single-cell Full-length Isoform Sequencing for Corneal Epithelium

2020 ◽  
Author(s):  
Ying-Feng Zheng ◽  
Zhi-Chao Chen ◽  
Zhuo-Xing Shi ◽  
Kun-Hua Hu ◽  
Jia-Yong Zhong ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Cost Effective ◽  
Pcr Primers ◽  
Cell Types ◽  
High Accuracy ◽  
Full Length ◽  
Pacbio Sequencing ◽  
Simple Method ◽  
Long Read

AbstractSingle-cell isoform sequencing can reveal transcriptomic dynamics in individual cells invisible to bulk- and single-cell RNA analysis based on short-read sequencing. However, current long-read single-cell sequencing technologies have been limited by low throughput and high error rate. Here we introduce HIT-scISOseq for high-throughput single-cell isoform sequencing. This method was made possible by full-length cDNA capture using biotinylated PCR primers, and by our novel library preparation procedure that combines head-to-tail concatemeric full-length cDNAs into a long SMRTbell insert for high-accuracy PacBio sequencing. HIT-scISOseq yields > 10 million high-accuracy full-length isoforms in a single PacBio Sequel II 8M SMRT Cell, providing > 8 times more data output than the standard single-cell isoform PacBio sequencing protocol. We exemplified HIT-scISOseq by first studying transcriptome profiles of 4,000 normal and 8,000 injured corneal epitheliums from cynomolgus monkeys. We constructed dynamic transcriptome landscapes of known and rare cell types, revealed novel isoforms, and identified injury-related splicing and switching events that are previously not accessible with low throughput isoform sequencing. HIT-scISOseq represents a high-throughput, cost-effective, and technically simple method to accelerate the burgeoning field of long-read single-cell transcriptomics.

scholarly journals Ultra-accurate microbial amplicon sequencing with synthetic long reads

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Benjamin J. Callahan ◽  
Dmitry Grinevich ◽  
Siddhartha Thakur ◽  
Michael A. Balamotis ◽  
Tuval Ben Yehezkel
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Full Length ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Strain Identification ◽  
Long Reads ◽  
Long Read

Abstract Background Out of the many pathogenic bacterial species that are known, only a fraction are readily identifiable directly from a complex microbial community using standard next generation DNA sequencing. Long-read sequencing offers the potential to identify a wider range of species and to differentiate between strains within a species, but attaining sufficient accuracy in complex metagenomes remains a challenge. Methods Here, we describe and analytically validate LoopSeq, a commercially available synthetic long-read (SLR) sequencing technology that generates highly accurate long reads from standard short reads. Results LoopSeq reads are sufficiently long and accurate to identify microbial genes and species directly from complex samples. LoopSeq perfectly recovered the full diversity of 16S rRNA genes from known strains in a synthetic microbial community. Full-length LoopSeq reads had a per-base error rate of 0.005%, which exceeds the accuracy reported for other long-read sequencing technologies. 18S-ITS and genomic sequencing of fungal and bacterial isolates confirmed that LoopSeq sequencing maintains that accuracy for reads up to 6 kb in length. LoopSeq full-length 16S rRNA reads could accurately classify organisms down to the species level in rinsate from retail meat samples, and could differentiate strains within species identified by the CDC as potential foodborne pathogens. Conclusions The order-of-magnitude improvement in length and accuracy over standard Illumina amplicon sequencing achieved with LoopSeq enables accurate species-level and strain identification from complex- to low-biomass microbiome samples. The ability to generate accurate and long microbiome sequencing reads using standard short read sequencers will accelerate the building of quality microbial sequence databases and removes a significant hurdle on the path to precision microbial genomics.

Sign in / Sign up

Export Citation Format

Share Document