Cell lineage analysis in human brain using endogenous retroelements

Reanalysis of Upton et al.'s data while filtering artifacts generates a consensus that retrotransposition occurs in developing brain but at a much lower rate consistent with prior single-cell studies.

“Reanalysis of their data while filtering these artifacts generates a consensus that retrotransposition does occur in developing brain but at a much lower rate consistent with prior single-cell studies (Evrony et al., 2012; Evrony et al., 2015), thereby constraining the range of possible functional roles for retrotransposition in the brain.”

Upton et al. single neurons have mean MAPD scores of 0.53 ± 0.16 (SD), compared to MAPD 0.18 ± 0.06 for MALBAC-amplified single cells from Zong et al. (2012) and MAPD 0.33 ± 0.06 for MDA-amplified single neurons from Evrony et al. (2015).

“Upton et al. single neurons have mean MAPD scores of 0.53 ± 0.16 (SD), compared to MAPD 0.18 ± 0.06 for MALBAC-amplified single cells from Zong et al. (2012) and MAPD 0.33 ± 0.06 for MDA-amplified single neurons from Evrony et al. (2015).”

These RC-seq somatic insertion rates are quite consistent with rates previously estimated by L1-IP in single cortical and caudate neurons (0.07 ± 0.15 SD; p = 0.54, ANOVA) and using single-neuron whole-genome sequencing (0.18 ± 0.47 SD; p = 0.37, ANOVA), suggesting a notable consensus by three methods confirming that somatic L1 insertions are present in human brain, but fewer than one per average.

“These RC-seq somatic insertion rates are quite consistent with rates previously estimated by L1 insertion profiling (L1-IP) in single cortical and caudate neurons (0.07 ± 0.15 (SD); p = 0.54, ANOVA) (Evrony et al., 2012), and using single-neuron whole-genome sequencing (0.18 ± 0.47 (SD); p = 0.37, ANOVA) (Evrony et al., 2015), suggesting a notable consensus by three methods confirming that somatic”

At ~500 kb genomic scales, MALBAC single cells from Zong et al. have high reproducibility and better uniformity of genome coverage than MDA.

“Of note, at these genomic scales, MALBAC single cells from Zong et al. have high reproducibility and better uniformity of genome coverage than MDA (Figure 4B; Figure 4—figure supplement 1A–B) (Evrony et al., 2015)”

Low-resolution genome coverage plots and statistics are minimally affected by increasing read depth >0.1x.

“confirming as in Evrony et al. (2015) that low-resolution genome coverage plots and statistics are minimally affected by increasing read depth > 0.1x.”

Power spectral density analysis confirmed better uniformity of MALBAC at large genomic scales (>30 kb) and better uniformity of MDA at small genomic scales (<30 kb).

“Power spectral density measuring amplification uniformity across all genomic scales confirmed better uniformity of MALBAC at large genomic scales (> 30 kb) and better uniformity of MDA at small genomic scales (< 30 kb) (Figure 4C) (Evrony et al., 2015; Zhang et al., 2015).”

16 single neurons were sequenced in Evrony et al. (2015), but the rate was estimated from the 14 single neurons that were selected randomly for sequencing.

“16 single neurons were sequenced in Evrony et al. (2015), but the rate was estimated from the 14 single neurons that were selected randomly for sequencing.”

The 2 remaining cells in which L1 #1 was detected (neurons 2 and 77) were excluded from the rate estimate because they were a priori chosen for whole-genome sequencing as positive controls known to harbor somatic L1 insertions previously detected by the L1-IP method in Evrony et al. (2012).

“The 2 remaining cells in which L1 #1 was detected (neurons 2 and 77) (Evrony et al., 2015) were excluded from the rate estimate, because they were a priori chosen for whole-genome sequencing as positive controls known to harbor somatic L1 insertions previously detected by the L1-IP method in Evrony et al. (2012).”

The calculated rate reflects the 2 of the 14 single neurons (neurons 6 and 18) that harbor the same L1 #2 clonal insertion.

“Therefore, the calculated rate reflects the 2 of the 14 single neurons (neurons 6 and 18) that harbor the same L1 #2 clonal insertion (Evrony et al., 2015).”

Single-cell sequencing can re-purpose LINE1 retrotransposition events for cell lineage analysis in the developing human brain.

“Interestingly, single-cell sequencing can re-purpose the LINE1 retrotransposition events for cell lineage analysis in the developing human brain (Evrony et al., 2015).”