Recent advances in DNA sequencing technologies based on single-molecule detection now enable determination of full-length transcript sequences and, thus, all protein sequences in a sample. Utilizing data from this exciting technology, we have constructed customized, full-length protein databases that offer unprecedented advantages in proteomics database searching. Protein inference from bottom-up proteomics data can now be conducted using the set of correct protein sequences actually expressed in the sample, meaning that peptide identifications can be understood in the context of their corresponding full-length protein sequences. And most importantly, novel peptides or proteins originating from variations in the genome or transcriptome can…
Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and…
The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans. Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building complete…
While advances in RNA sequencing methods have accelerated our understanding of the human transcriptome, isoform discovery remains a challenge because short read lengths require complicated assembly algorithms to infer the contiguity of full-length transcripts. With PacBio’s long reads, one can now sequence full-length transcript isoforms up to 10 kb. The PacBio Iso- Seq protocol produces reads that originate from independent observations of single molecules, meaning no assembly is needed. Here, we sequenced the transcriptome of the human MCF-7 breast cancer cell line using the Clontech SMARTer® cDNA preparation kit and the PacBio RS II. Using PacBio Iso-Seq bioinformatics software, we…
Advances in RNA sequencing have accelerated our understanding of the transcriptome, however isoform discovery remains challenging due to short read lengths. The Iso-Seq Application provides a new alternative to sequence full-length cDNA libraries using long reads from the PacBio RS II. Identification of long and often rare isoforms is demonstrated with rat heart and lung RNA prepared using the Clontech® SMARTer® cDNA preparation kit, followed by agarose-gel size selection in fractions of 1-2 kb, 2-3 kb and 3-6 kb. For each tissue, 1.8 and 1.2 million reads were obtained from 32 and 26 SMRT Cells, respectively. Filtering for reads with…
In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey…
PacBio bioinformatician, Elizabeth Tseng, reviews the bioinformatics strategies utilizing PacBio long-read sequencing data for isoform sequencing for full-length transcript sequencing without assembly.
The majority of human genes are alternatively spliced, making it possible for most genes to generate multiple proteins. The process of alternative splicing is highly regulated in a developmental-stage and tissue-specific manner. Perturbations in the regulation of these events can lead to disease in humans (1). Alternative splicing has been shown to play a role in human cancer, muscular dystrophy, Alzheimer’s, and many other diseases. Understanding these diseases requires knowing the full complement of mRNA isoforms. Microarrays and high-throughput cDNA sequencing have become highly successful tools for studying transcriptomes, however these technologies only provide small fragments of transcripts and building…
In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties and functions. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be understated. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for…