N procedures may be insufficient because SHM can affect the efficiency of PCR amplification and not every single somatic variant can realistically be captured in the calibrators. Alternatively, one could start from RNA and use primers that are in the constant region to circumvent mutation. But with RNA from bulk populations, one has to correct for differences in transcript abundance in different cell types or perform amplifications on sorted or single cells. In addition to using calibrators or some other normalization technique, one can generate replicate sequencing datasets from the same sample [60]. By performing multiple replicate amplifications from independent DNA aliquots of the same sample, one can determine the maximal expected clonal overlap within a given sample, determine which clones are reliably identified in the replicates (including the reproducibility of their copy Y-27632 mechanism of action number rankings) and, with adequate sampling, perform a rarefaction analysis to empirically determine which clones can be identified reliably [61]. A final method to assess the relative extent of clonal expansion is to count the number of unique sequences in a given clone, counting every variant of mutant sequence in a clone only once. While this is not a direct measurement of all expansion as it depends on mutation, it is nonetheless quite reliable, when mutations are present.VH usage on the basis of clone copy numbers. A comparison of the VH usage of clonotypes (where each clone is only counted once) with VH usage with total copies provides insights into the effects of high copy number clones on the repertoire. VH usage is frequently shown as a heat map, and various types of clustering algorithms can be used to compare VH usage in different sequencing libraries, similar to what investigators use for microarray data. Another general metric of repertoire skewing is the size distribution of antibody CDR3 lengths. In a order LM22A-4 diverse repertoire, we expect the distribution of the CDR3 sizes to resemble a truncated and discretized Gaussian distribution [62]. If the size distribution of CDR3s in the repertoire exhibits kurtosis or skewing, this can be an indication of an unexpected over-abundance of clones undergoing abnormal development or perhaps reacting to a specific antigen. For instance, antibody sequences with short CDR3 lengths may derive from B1 B cells, which develop in fetal life and tend to have fewer N additions because the enzyme that creates N additions (terminal deoxynucleotidyl transferase) is less active during fetal life [40]. Conversely, human antibody sequences with long CDR3 lengths may have an increased frequency of JH6 usage (JH6 has a series of tyrosine residues at the 50 end that contribute towards the CDR3 length, as was observed in early stage B-cell precursors [63]). Another potential molecular mechanism for CDR3 elongation is VH replacement. In VH replacement, an upstream VH gene invades into a pre-existing VDJ rearrangement on the same allele [64,65]. VH replacement can elongate the CDR3, because the invasion occurs into a cryptic heptamer sequence that is upstream of the V junction. Thus, in some cases, the 30 end of the VH gene in the replaced allele not only contains the original V junction, but also a few nucleotides from the 30 end of the VH gene itself, the so-called VH replacement `footprint’ [66].rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 370:7. Distinguishing somatic mutation from sequencing errorsAnother general metri.N procedures may be insufficient because SHM can affect the efficiency of PCR amplification and not every single somatic variant can realistically be captured in the calibrators. Alternatively, one could start from RNA and use primers that are in the constant region to circumvent mutation. But with RNA from bulk populations, one has to correct for differences in transcript abundance in different cell types or perform amplifications on sorted or single cells. In addition to using calibrators or some other normalization technique, one can generate replicate sequencing datasets from the same sample [60]. By performing multiple replicate amplifications from independent DNA aliquots of the same sample, one can determine the maximal expected clonal overlap within a given sample, determine which clones are reliably identified in the replicates (including the reproducibility of their copy number rankings) and, with adequate sampling, perform a rarefaction analysis to empirically determine which clones can be identified reliably [61]. A final method to assess the relative extent of clonal expansion is to count the number of unique sequences in a given clone, counting every variant of mutant sequence in a clone only once. While this is not a direct measurement of all expansion as it depends on mutation, it is nonetheless quite reliable, when mutations are present.VH usage on the basis of clone copy numbers. A comparison of the VH usage of clonotypes (where each clone is only counted once) with VH usage with total copies provides insights into the effects of high copy number clones on the repertoire. VH usage is frequently shown as a heat map, and various types of clustering algorithms can be used to compare VH usage in different sequencing libraries, similar to what investigators use for microarray data. Another general metric of repertoire skewing is the size distribution of antibody CDR3 lengths. In a diverse repertoire, we expect the distribution of the CDR3 sizes to resemble a truncated and discretized Gaussian distribution [62]. If the size distribution of CDR3s in the repertoire exhibits kurtosis or skewing, this can be an indication of an unexpected over-abundance of clones undergoing abnormal development or perhaps reacting to a specific antigen. For instance, antibody sequences with short CDR3 lengths may derive from B1 B cells, which develop in fetal life and tend to have fewer N additions because the enzyme that creates N additions (terminal deoxynucleotidyl transferase) is less active during fetal life [40]. Conversely, human antibody sequences with long CDR3 lengths may have an increased frequency of JH6 usage (JH6 has a series of tyrosine residues at the 50 end that contribute towards the CDR3 length, as was observed in early stage B-cell precursors [63]). Another potential molecular mechanism for CDR3 elongation is VH replacement. In VH replacement, an upstream VH gene invades into a pre-existing VDJ rearrangement on the same allele [64,65]. VH replacement can elongate the CDR3, because the invasion occurs into a cryptic heptamer sequence that is upstream of the V junction. Thus, in some cases, the 30 end of the VH gene in the replaced allele not only contains the original V junction, but also a few nucleotides from the 30 end of the VH gene itself, the so-called VH replacement `footprint’ [66].rstb.royalsocietypublishing.org Phil. Trans. R. Soc. B 370:7. Distinguishing somatic mutation from sequencing errorsAnother general metri.
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BET Bromodomain Inhibitor
