Whole-Genome Sequencing of Multiple Myeloma

Informed consent from MM patients was obtained in line with the Declaration of Helsinki. DNA was extracted from bone marrow aspirate (tumor) and blood (normal). WGS libraries (370-410 bp inserts) and WES libraries (200-350 bp inserts) were constructed and sequenced on an Illumina GA-II sequencer using 101 and 76 bp paired-end reads, respectively. Sequencing reads were procesed with the Firehose pipeline, identifying somatic point mutations, indels, and other structural chromosomal rearrangements. Structural rearrangements affecting protein-coding regions were then subjected to manual review to exclude alignment artifacts. True positive mutation rates were estimated by Sequenom mass spectrometry genotyping of randomly selected mutations. HOXA9 shRNAs were introduced into MM cell lines using lentiviral infection using standard methods.
A complete description of the materials and methods are provided in the Supplementary Information.

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Chapman M.A., Lawrence M.S., Keats J.J., Cibulskis K., Sougnez C., Schinzel A.C., Harview C.L., Brunet J.P., Ahmann G.J., Adli M., Anderson K.C., Ardlie K.G., Auclair D., Baker A., Bergsagel P.L., Bernstein B.E., Drier Y., Fonseca R., Gabriel S.B., Hofmeister C.C., Jagannath S., Jakubowiak A.J., Krishnan A., Levy J., Liefeld T., Lonial S., Mahan S., Mfuko B., Monti S., Perkins L.M., Onofrio R., Pugh T.J., Vincent Rajkumar S., Ramos A.H., Siegel D.S., Sivachenko A., Trudel S., Vij R., Voet D., Winckler W., Zimmerman T., Carpten J., Trent J., Hahn W.C., Garraway L.A., Meyerson M., Lander E.S., Getz G, & Golub T.R. (2011). Initial genome sequencing and analysis of multiple myeloma. Nature, 471(7339), 467-472.

Publication 2011

Blood Bone marrow Cell lines Chromosomal Ga ii Hoxa9 Indels Infection Mass spectrometry Mutations Patients Point mutations Protein coding regions Rearrangements Shrnas Somatic Tumor

Corresponding Organization :

Other organizations : Broad Institute, WinnMed, Mayo Clinic, Harvard University Press, Multiple Myeloma Research Foundation, Translational Genomics Research Institute, Howard Hughes Medical Institute, Weizmann Institute of Science, The Ohio State University, Catholic Medical Center, Ann Arbor Center for Independent Living, University of Michigan–Ann Arbor, City Of Hope National Medical Center, Emory University, Rutgers, The State University of New Jersey, Washington University in St. Louis, University of Chicago, Massachusetts Institute of Technology, Dana-Farber Cancer Institute

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Protocol cited in 113 other protocols

Variable analysis

independent variables

Introducing HOXA9 shRNAs into MM cell lines using lentiviral infection

dependent variables

Somatic point mutations
Indels
Structural chromosomal rearrangements

control variables

Bone marrow aspirate (tumor) and blood (normal) samples
Sequencing read processing using the Firehose pipeline
Manual review of structural rearrangements affecting protein-coding regions to exclude alignment artifacts
Sequenom mass spectrometry genotyping of randomly selected mutations to estimate true positive mutation rates

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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