Reverse Transcription Errors and RNA–DNA Differences at Short Tandem Repeats

Abstract

Transcript variation has important implications for organismal function in health and disease. Most transcriptome studies focus on assessing variation in gene expression levels and isoform representation. Variation at the level of transcript sequence is caused by RNA editing and transcription errors, and leads to nongenetically encoded transcript variants, or RNA–DNA differences (RDDs). Such variation has been understudied, in part because its detection is obscured by reverse transcription (RT) and sequencing errors. It has only been evaluated for intertranscript base substitution differences. Here, we investigated transcript sequence variation for short tandem repeats (STRs). We developed the first maximum-likelihood estimator (MLE) to infer RT error and RDD rates, taking next generation sequencing error rates into account. Using the MLE, we empirically evaluated RT error and RDD rates for STRs in a large-scale DNA and RNA replicated sequencing experiment conducted in a primate species. The RT error rates increased exponentially with STR length and were biased toward expansions. The RDD rates were approximately 1 order of magnitude lower than the RT error rates. The RT error rates estimated with the MLE from a primate data set were concordant with those estimated with an independent method, barcoded RNA sequencing, from a Caenorhabditis elegans data set. Our results have important implications for medical genomics, as STR allelic variation is associated with >40 diseases. STR nonallelic transcript variation can also contribute to disease phenotype. The MLE and empirical rates presented here can be used to evaluate the probability of disease-associated transcripts arising due to RDD.

IsoCon: Deciphering highly similar multigene family transcripts from Iso-Seq data

“…Several experimental improvements can be incorporated into IsoCon as well. First, to eliminate reverse transcriptase and sequencing errors, two replicate cDNA libraries can be prepared from the same sample 56 . Any differences in transcript sequences between the two libraries will be consistent with such errors.…”

Relatively semi-conservative replication and a folded slippage model for simple sequence repeats

“…However, if the remained high percentage of short repeats is produced during replication process as described above, it certainly makes the base numbers of replication strand to be unequal to those of template strand, with one or several nucleotides/motifs being repeated and more than that in template strand. In vitro experiments also revealed the presence of repeats during DNA replication, a nd the nascent 6 replication chain has a base increase (Doublié et al, 1998;Fungtammasan et al, 2015;Fungtammasan et al, 2016;Kiefer et al, 1998). And in this case, the replication process is possibly relatively semi-conservative and could be described as the following formula:…”

“…However, there are two manners for the repeat sequences contraction, one is above mentioned the template chain folds on outside, another is also above stated general mutations; the high content of the repeat sequence is still in a stable state in the genome of each species, suggesting that the possibility of repeat expansion should be higher than repeat contraction. And many reports also suggest that there is a higher possibility of repeat expansion than repeat contraction (Fungtammasan et al, 2015;Fungtammasan et al, 2016;Neil et al, 2018).…”

Relatively semi-conservative replication and a folded slippage model for simple sequence repeats

“…However, if the remained high percentage of short repeats is produced during replication process as described above, it certainly makes the base numbers of replication strand to be unequal to those of template strand, with one or several nucleotides/motifs being repeated and more than that in template strand. In vitro experiments also revealed the presence of repeats during DNA replication, and the nascent replication chain has a base increase [27,37,38,49]. And in this case, the replication process is possibly relatively semi-conservative and could be described as the following formula:…”

“…However, there are two manners for the repeat sequences contraction, one is above mentioned the template chain folds on outside, another is also above stated general mutations; the high content of the repeat sequence is still in a stable state in the genome of each species, suggesting that the possibility of repeat expansion should be higher than repeat contraction. And many reports also suggest that there is a higher possibility of repeat expansion than repeat contraction [27,49,67].…”