Long-range massively parallel mate pair sequencing detects distinct mutations and similar patterns of structural mutability in two breast cancer cell lines

Hampton, Oliver A.; Miller, Christopher A.; Koriabine, Maxim; Li, Jian; Hollander, Petra den; Carbone, Lucia; Nefedov, Mikhail; Hallers, Boudewijn F.H. ten; Lee, Adrian V.; Jong, Pieter J. de; Milosavljevic, Aleksandar

doi:10.1016/j.cancergen.2011.07.009

Cited by 17 publications

(18 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this respect, Fosills are similar to Fosmid ''diTags'' (Hampton et al 2011). The principal advantage of our method is that it allows much longer sequencing reads (up to 2 3 101 bases in the current study, but even longer reads are possible), whereas the EcoP15I digest strictly limits the diTags to 2 3 26 bases.…”

Section: Discussionmentioning

confidence: 92%

See 1 more Smart Citation

Paired-end sequencing of Fosmid libraries by Illumina

Williams

Tabbaa

et al. 2012

Genome Res.

View full text Add to dashboard Cite

Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the Fosmid-or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments without physical size selection and is compatible with standard and multiplex paired-end Illumina sequencing. To excise the bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them. Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for paired-end sequencing with 101-base reads. The yield of unique Fosmid-sized jumps is sufficiently high, and the background of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing based draft assembly.

show abstract

Section: Discussionmentioning

confidence: 92%

“…To close this technology gap, we and others have taken a hybrid approach wherein Fosmid libraries are constructed first and then converted to Fosmid-size jumps in vitro (Gnerre et al 2011;Hampton et al 2011). …”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Paired-end sequencing of Fosmid libraries by Illumina

Williams

Tabbaa

et al. 2012

Genome Res.

View full text Add to dashboard Cite

show abstract

“…Much larger inserts can be produced by circularizing large DNA fragments of up to 10 kb (Fullwood et al 2009;Hillmer et al 2011), and subsequent isolation of a short fragment that contains both ends (mate pairs). Large-insert and fosmid mate-pair libraries offer several attractive features that make them well-suited for analysis of structural variation (Raphael et al 2003;International Human Genome Sequencing Consortium 2004;Tuzun et al 2005;Kidd et al 2008;Hampton et al 2011;Williams et al 2012). First, mate pairs inherently capture genomic structure in that discordantly aligning mate-pair reads occur at sites of genomic rearrangements, exposing underlying lesions (Supplemental Fig.…”

mentioning

confidence: 99%

Discovery of recurrent structural variants in nasopharyngeal carcinoma

et al. 2013

View full text Add to dashboard Cite

We present the discovery of genes recurrently involved in structural variation in nasopharyngeal carcinoma (NPC) and the identification of a novel type of somatic structural variant. We identified the variants with high complexity mate-pair libraries and a novel computational algorithm specifically designed for tumor-normal comparisons, SMASH. SMASH combines signals from split reads and mate-pair discordance to detect somatic structural variants. We demonstrate a >90% validation rate and a breakpoint reconstruction accuracy of 3 bp by Sanger sequencing. Our approach identified three in-frame gene fusions (YAP1-MAML2, PTPLB-RSRC1, and SP3-PTK2) that had strong levels of expression in corresponding NPC tissues. We found two cases of a novel type of structural variant, which we call ''coupled inversion,'' one of which produced the YAP1-MAML2 fusion. To investigate whether the identified fusion genes are recurrent, we performed fluorescent in situ hybridization (FISH) to screen 196 independent NPC cases. We observed recurrent rearrangements of MAML2 (three cases), PTK2 (six cases), and SP3 (two cases), corresponding to a combined rate of structural variation recurrence of 6% among tested NPC tissues.

show abstract

“…In the MCF-7 cell-line samples, five fusion events were found: two fusions spanning RPS6KB1-VMP1, ARFGEF2-SULF2, DEPDC1B-ELOVL7, and GRB2-SUMO2 (Supplemental Figure S7), most of which have previously been reported. 19,20 In the spiked blood sample, all of these five fusion events were detected, and the results were conclusive irrespective of fixation (Figure 7 …”

Section: Identification Of Tumor Cell Signals In Spiked Blood Samplesmentioning

confidence: 80%

Toward Rare Blood Cell Preservation for RNA Sequencing

Vicković

Ahmadian

Lewensohn

et al. 2015

The Journal of Molecular Diagnostics

View full text Add to dashboard Cite

Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues-a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and long-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis.

show abstract

Long-range massively parallel mate pair sequencing detects distinct mutations and similar patterns of structural mutability in two breast cancer cell lines

Cited by 17 publications

References 56 publications

Paired-end sequencing of Fosmid libraries by Illumina

Paired-end sequencing of Fosmid libraries by Illumina

Discovery of recurrent structural variants in nasopharyngeal carcinoma

Toward Rare Blood Cell Preservation for RNA Sequencing

Contact Info

Product

Resources

About