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Highly efficient genomic enrichment of human exonic sequences through short-fragment libraries

ABSTRACT: Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we have explored the use of short fragment libraries (85-110bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. These short fragment libraries were enriched for 1.69 Mb of exonic sequences, using custom 244K microarrays, and sequenced using AB/SOLiD. High enrichment specificity (60 – 75%) was obtained at 67-213x average coverage, with 77-92% and 90-98% of targeted regions covered with more than 25% and 10% of the average coverage, respectively. As a more appropriate measure of the evenness of coverage, which is relatively independent of sequencing depth, we introduce the evenness of coverage parameter E. E values up to 75% were achieved. To verify the accuracy of SNP/mutation detection we evaluated 384 known non-reference SNPs in the targeted regions. At ~ 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls while 3.6% of targeted regions were marked as unsurveyed. A total of 1197 new variants were detected. Verification revealed only 8 false positive calls, resulting in an overall false positive rate of less than 1 per ~200,000 bp (0.0005%, equivalent to an overall phred score of 55).

ORGANISM(S): Homo sapiens

PROVIDER: GSE18542 | GEO | 2010/02/01

SECONDARY ACCESSION(S): PRJNA121347

REPOSITORIES: GEO

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Project description:The sensitivity of single-cell proteomics (SCP) has increased dramatically in recent years due to advances in experimental design, sample preparation, separations and mass spectrometry instrumentation. However, further increasing the sensitivity of SCP methods and instrumentation will enable the study of proteins within single cells that are expressed at copy numbers too small to be measured by current methods. Here we further increase SCP sensitivity by combining efficient nanoPOTS sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to extend the achievable proteome coverage for single cells to >3,000 for single-cell data analyses. WWA is based on data-dependent acquisition (DDA) but employs larger precursor isolation windows to intentionally co-isolate and co-fragment additional precursors along with the selected precursor. The resulting chimeric MS2 spectra are then resolved using the CHIMERYS search engine within Proteome Discoverer 3.0. Compared to standard DDA workflows, WWA employing isolation windows of 8-12 Th increases peptide and proteome coverage by ~28% and ~39%, respectively. For a 40 min LC gradient operated at ~15 nL/min, we identified an average of 2,150 proteins per single-cell-sized aliquots of protein digest directly from MS2 spectra, which increased to an average 3,524 proteins including proteins identified with MS1-level feature matching. Reducing the active gradient to 20 min resulted in only a 10% decrease in proteome coverage. We also compared performance of WWA with DIA underperformed relative to WWA in terms of proteome coverage, especially with faster separations. Average proteome coverage for single HeLa and K562 cells was respectively 1,758 and 1,642 based on MS2 identifications with 1% false discovery rate and 3042 and 2891 with MS1 feature matching. As such, WWA combined with efficient sample preparation and rapid separations extends the depths of the proteome that can be studied at the single-cell level.

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Highly efficient genomic enrichment of human exonic sequences through short-fragment libraries

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