title: Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.
abstract: BACKGROUND
  Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.
  
  
  RESULTS
  We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.
  
  
  CONCLUSIONS
  PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.
authors.affiliation: Critical Care Medicine Department, Clinical Center, Frederick, MD USA.
authors.forename: Ma
authors.initials: M
authors.lastname: Liang
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Castle
authors.initials: C
authors.lastname: Raley
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Critical Care Medicine Department, Clinical Center, Frederick, MD USA.
authors.forename: Geetha
authors.initials: G
authors.lastname: Kutty
authors.affiliation: Critical Care Medicine Department, Clinical Center, Frederick, MD USA.
authors.forename: Emile
authors.initials: E
authors.lastname: Gogineni
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Brad T
authors.initials: BT
authors.lastname: Sherman
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Sun
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Xiongfong
authors.initials: X
authors.lastname: Chen
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Skelly
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Kristine
authors.initials: K
authors.lastname: Jones
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Stephens
authors.affiliation: Center of Information Technology, National Institutes of Health (NIH), Bethesda, MD USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Center of Information Technology, National Institutes of Health (NIH), Bethesda, MD USA.
authors.forename: William
authors.initials: W
authors.lastname: Lau
authors.affiliation: Center of Information Technology, National Institutes of Health (NIH), Bethesda, MD USA.
authors.forename: Calvin
authors.initials: C
authors.lastname: Johnson
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Tomozumi
authors.initials: T
authors.lastname: Imamichi
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Minkang
authors.initials: M
authors.lastname: Jiang
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Robin
authors.initials: R
authors.lastname: Dewar
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Lempicki
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.
authors.forename: Bao
authors.initials: B
authors.lastname: Tran
authors.affiliation: Critical Care Medicine Department, Clinical Center, Frederick, MD USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Kovacs
authors.affiliation: Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.|Current Affiliation: National Cancer Institute, NIH, Bethesda, MD USA.
authors.forename: Da Wei
authors.initials: DW
authors.lastname: Huang
keywords: Bioinformatics analysis; Gene isoforms; Major surface glycoprotein; NGS; PacBio; Pneumocystis; Repetitive sequences; Uclust