Human X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity. We investigated whether the sequence homology between the X and Y chromosomes affects the alignment of RNA-Seq reads and estimates of differential expression. We tested the effects of using reference genomes and reference transcriptomes informed by the sex chromosome complement of the sample’s genome on the measurements of RNA-Seq abundance and sex differences in expression. The default genome includes the entire human reference genome (GRCh38), including the entire sequence of the X and Y chromosomes. We created two sex chromosome complement informed reference genomes. One sex chromosome complement informed reference genome was used for samples that lacked a Y chromosome; for this reference genome version, we hard-masked the entire Y chromosome. For the other sex chromosome complement informed reference genome, to be used for samples with a Y chromosome, we hard-masked only the pseudoautosomal regions of the Y chromosome, because these regions are duplicated identically in the reference genome on the X chromosome. We analyzed the transcript abundance in the whole blood, brain cortex, breast, liver, and thyroid tissues from 20 genetic female (46, XX) and 20 genetic male (46, XY) samples. Each sample was aligned twice: once to the default reference genome and then independently aligned to a reference genome informed by the sex chromosome complement of the sample, repeated using two different read aligners, HISAT and STAR. We then quantified sex differences in gene expression using featureCounts to get the raw count estimates followed by Limma/Voom for normalization and differential expression. We additionally created sex chromosome complement informed transcriptome references for use in pseudo-alignment using Salmon. Transcript abundance was quantified twice for each sample: once to the default target transcripts and then independently to target transcripts informed by the sex chromosome complement of the sample. We show that regardless of the choice of the read aligner, using an alignment protocol informed by the sex chromosome complement of the sample results in higher expression estimates on the pseudoautosomal regions of the X chromosome in both genetic male and genetic female samples, as well as an increased number of unique genes being called as differentially expressed between the sexes. We additionally show that using a pseudo-alignment approach informed on the sex chromosome complement of the sample eliminates Y-linked expression in female XX samples.

中文翻译：

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参考基因组和转录组由样本的性染色体补体提供信息，提高了从 RNA-Seq 数据检测基因表达性别差异的能力。

人类 X 和 Y 染色体共享进化起源，因此具有序列相似性。我们研究了 X 和 Y 染色体之间的序列同源性是否影响 RNA-Seq 读数的比对和差异表达的估计。我们测试了使用参考基因​​组和参考转录组的影响，这些参考基因组和参考转录组由样本基因组的性染色体补体提供信息，对 RNA-Seq 丰度和表达中的性别差异的测量的影响。默认基因组包括整个人类参考基因组 (GRCh38)，包括 X 和 Y 染色体的整个序列。我们创建了两个性染色体补充知情参考基因组。一个性染色体补充告知参考基因组用于缺乏 Y 染色体的样本；对于这个参考基因组版本，我们硬屏蔽了整个 Y 染色体。对于其他性染色体补充告知参考基因组，要用于带有 Y 染色体的样本，我们仅硬掩蔽了 Y 染色体的假常染色体区域，因为这些区域在 X 染色体上的参考基因组中重复相同。我们分析了来自 20 个遗传女性 (46, XX) 和 20 个遗传男性 (46, XY) 样本的全血、脑皮质、乳腺、肝脏和甲状腺组织中的转录丰度。每个样本都比对了两次：一次与默认参考基因组比对，然后与由样本的性染色体互补告知的参考基因组独立比对，使用两个不同的读取比对器 HISAT 和 STAR 重复。然后我们使用 featureCounts 量化基因表达的性别差异以获得原始计数估计值，然后使用 Limma/Voom 进行标准化和差异表达。我们还创建了性染色体补充信息转录组参考，用于使用 Salmon 进行伪比对。每个样本的转录本丰度被量化两次：一次是默认的目标转录本，然后独立到由样本的性染色体补体通知的目标转录本。我们表明，无论 read aligner 的选择如何，使用由样本的性染色体补体通知的对齐协议都会导致对基因男性和基因女性样本中 X 染色体假常染色体区域的更高表达估计，以及越来越多的独特基因被称为在两性之间差异表达。我们还表明，使用基于样本的性染色体补体的伪比对方法消除了女性 XX 样本中的 Y 连锁表达。