Introduction to CyTOF

BY IN Notes, Uncategorized NO COMMENTS YET , , , ,

Motivation: Characterizing large quantities of single cells is important to address clinically and biologically relevant questions. Traditionally, bulk measurements have been used to characterize the functional states of diseased vs. normal immune cells, livers, brains, and tissues or organs of interest to identify various interesting mechanistic differences. However, these bulk measurements average information across many

CONTINUE READING …

Genome amplification approaches: DOP-PCR, MDA, MALBAC

BY IN Notes NO COMMENTS YET , , , ,

DOP-PCR = degenerate oligonucleotide priming polymerase chain reaction Method Overview: In the first cycling stage (5-8 cycles), low-temperature annealing and extension occur at many binding sites in the genome and become tagged with the DOP primer. In the second cycling stage (>25 cycles), annealing temperature is raised, increasing priming specificity during amplification of the tagged

CONTINUE READING …

SNP vs. SNV vs. Mutation

BY IN Notes 2 COMMENTS ,

SNP = single nucleotide polymorphism. As its name suggests, a SNP is a DNA change at the single nucleotide level. By virtue of being a polymorphism, a SNP must occur commonly within a population, with “common” typically being understood as having a minor allele frequency of >1%. The commonality of SNPs generally implies that SNPs

CONTINUE READING …

RPKM and FPKM explained

BY IN Notes 17 COMMENTS , , ,

RNA-Seq provides quantitative approximations of the abundance of target transcripts in the form of counts. However, these counts must be normalized to remove technical biases inherent in the preparation steps for RNA-Seq, in particular the length of the RNA species and the sequencing depth of a sample. For example, expectedly, deeper sequencing results in higher

CONTINUE READING …

Improving RNA-Seq expression estimates by correcting for fragment bias

BY IN Journal Club, RNA-Seq NO COMMENTS YET , , , ,

Preparation steps for RNA-Seq lead to both positional bias, whereby fragments are preferential located towards either the beginning or end of transcripts, and sequence-specific bias, whereby the sequence surrounding the beginning or end of potential fragments affects their likelihood of being selected for sequencing, in sequenced fragments. If such biases are not corrected for, expression

CONTINUE READING …