A large genome center's improvements to the Illumina sequencing system

Quail, Michael A.; Kozarewa, Iwanka; Smith, Frances J.D.; Scally, Aylwyn; Stephens, Philip J.; Durbin, Richard; Swerdlow, Harold; Turner, Daniel J.

doi:10.1038/nmeth.1270

Cited by 640 publications

(559 citation statements)

References 11 publications

Supporting

Mentioning

552

Contrasting

Unclassified

Order By: Relevance

“…Overall, the data suggest similar or better reproducibility than previously reported for microarray-based sequence capture Hodges et al 2007;Okou et al 2007;Bau et al 2009). Importantly, analysis of the uniqueness of obtained read pairs revealed that more than 98% for both runs, were unique, which is higher than previously reported for standard Illumina GAII sequencing without any enrichment method (Quail et al 2008). This clearly shows that no detectable library representation bias has been introduced during the HybSelect process that would compromise the information value of obtained reads.…”

Section: Capture Of Cancer-related Genesmentioning

confidence: 51%

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Summerer¹,

Wu²,

Haase³

et al. 2009

Genome Res.

View full text Add to dashboard Cite

The lack of efficient high-throughput methods for enrichment of specific sequences from genomic DNA represents a key bottleneck in exploiting the enormous potential of next-generation sequencers. Such methods would allow for a systematic and targeted analysis of relevant genomic regions. Recent studies reported sequence enrichment using a hybridization step to specific DNA capture probes as a possible solution to the problem. However, so far no method has provided sufficient depths of coverage for reliable base calling over the entire target regions. We report a strategy to multiply the enrichment performance and consequently improve depth and breadth of coverage for desired target sequences by applying two iterative cycles of hybridization with microfluidic Geniom biochips. Using this strategy, we enriched and then sequenced the cancer-related genes BRCA1 and TP53 and a set of 1000 individual dbSNP regions of 500 bp using Illumina technology. We achieved overall enrichment factors of up to 1062-fold and average coverage depths of 470-fold. Combined with high coverage uniformity, this resulted in nearly complete consensus coverages with >86% of target region covered at 20-fold or higher. Analysis of SNP calling accuracies after enrichment revealed excellent concordance, with the reference sequence closely mirroring the previously reported performance of Illumina sequencing conducted without sequence enrichment.

show abstract

Section: Capture Of Cancer-related Genesmentioning

confidence: 51%

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Summerer¹,

Wu²,

Haase³

et al. 2009

Genome Res.

View full text Add to dashboard Cite

show abstract

“…The 39 end of these primers is modified with phosphorothioate, preventing degradation of the primer by error-correcting polymerases (Quail et al 2008). The amplified library was flourometrically quantified on a Qubit flourimeter (Invitrogen) and sequenced on an Illumina Genome Analyzer (Princeton Microarray Facility, http://www.genomics.princeton.edu/ microarray/) using the standard Illumina protocols.…”

Section: Production Of the Bar-coded Librarymentioning

confidence: 99%

Multiplexed shotgun genotyping for rapid and efficient genetic mapping

et al. 2011

View full text Add to dashboard Cite

We present a new approach to genotyping based on multiplexed shotgun sequencing that can identify recombination breakpoints in a large number of individuals simultaneously at a resolution sufficient for most mapping purposes, such as quantitative trait locus (QTL) mapping and mapping of induced mutations. We first describe a simple library construction protocol that uses just 10 ng of genomic DNA per individual and makes the approach accessible to any laboratory with standard molecular biology equipment. Sequencing this library results in a large number of sequence reads widely distributed across the genomes of multiplexed bar-coded individuals. We develop a Hidden Markov Model to estimate ancestry at all genomic locations in all individuals using these data. We demonstrate the utility of the approach by mapping a dominant marker allele in D. simulans to within 105 kb of its true position using 96 F1-backcross individuals genotyped in a single lane on an Illumina Genome Analyzer. We further demonstrate the utility of our method by genetically mapping more than 400 previously unassembled D. simulans contigs to linkage groups and by evaluating the quality of targeted introgression lines. At this level of multiplexing and divergence between strains, our method allows estimation of recombination breakpoints to a median of 38-kb intervals. Our analysis suggests that higher levels of multiplexing and/or use of strains with lower levels of divergence are practicable.

show abstract

“…This was postulated by Quail et al (2008) as a means to reduce the inclusion of artifactual chimeric transcripts that are composed of two cDNA fragments into the sequencing library. We also integrated the use of T4 ligase (Enzymatics Inc.) to improve the efficiency of adapter ligation.…”

Section: Discovery Of Non-ets Gene Fusions In Prostate Cancermentioning

confidence: 99%

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing

Pflueger

Terry²,

Sboner³

et al. 2010

Genome Res.

184

156

View full text Add to dashboard Cite

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date, little is known about the presence of non-ETS fusion events in prostate cancer. We used next-generation transcriptome sequencing (RNA-seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and four involving non-ETS genes such as CDKN1A (p21), CD9, and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency, but, interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement-prone phenotype.

show abstract

A large genome center's improvements to the Illumina sequencing system

Cited by 640 publications

References 11 publications

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Microarray-based multicycle-enrichment of genomic subsets for targeted next-generation sequencing

Multiplexed shotgun genotyping for rapid and efficient genetic mapping

Discovery of non-ETS gene fusions in human prostate cancer using next-generation RNA sequencing

Contact Info

Product

Resources

About