Abstract：
BACKGROUND: High-throughput single-cell RNA sequencing (scRNA-seq) is widely used in spermatogenesis. However, it only reveals short reads in germ and somatic cells, limiting the discovery of novel transcripts and genes.
OBJECTIVE: This study shows the long-read transcriptional landscape of spermatogenesis in obstructive azoospermia (OA) and Sertoli cell-only patients.
METHODS: Single cells were isolated from testicular biopsies of OA and non-obstructive azoospermia (NOA) patients. Cell culture was identified by comparing PacBio long-read single-cell sequencing (OA n = 3, NOA n = 3) with short-read scRNA-seq (OA n = 6, NOA n = 6). Ten germ cell types and eight somatic cell types were classified based on known markers.
METHODS: PacBio long-read single-cell sequencing, short-read scRNA-seq, polymerase chain reaction.
RESULTS: A total of 130 426 long-read transcripts (100 517 novel transcripts and 29 909 known transcripts) and 49 508 long-read transcripts (26 002 novel transcripts and 23 506 known transcripts) have been detected in OA and NOA patients, respectively. Moreover, 36 373 and 1642 new genes are identified in OA and NOA patients, respectively. Importantly, specific expressions of long-read transcripts were detected in germ and stomatic cells during normal spermatogenesis.
CONCLUSIONS: We have identified total full-length transcripts in OA and NOA, and new genes were found. Furthermore, specific expressed full-length transcripts were detected, and the genomic structure of transcripts was mapped in different cell types. These findings may provide valuable information on human spermatogenesis and the treatment of male infertility.
OBJECTIVE: This study shows the long-read transcriptional landscape of spermatogenesis in obstructive azoospermia (OA) and Sertoli cell-only patients.
METHODS: Single cells were isolated from testicular biopsies of OA and non-obstructive azoospermia (NOA) patients. Cell culture was identified by comparing PacBio long-read single-cell sequencing (OA n = 3, NOA n = 3) with short-read scRNA-seq (OA n = 6, NOA n = 6). Ten germ cell types and eight somatic cell types were classified based on known markers.
METHODS: PacBio long-read single-cell sequencing, short-read scRNA-seq, polymerase chain reaction.
RESULTS: A total of 130 426 long-read transcripts (100 517 novel transcripts and 29 909 known transcripts) and 49 508 long-read transcripts (26 002 novel transcripts and 23 506 known transcripts) have been detected in OA and NOA patients, respectively. Moreover, 36 373 and 1642 new genes are identified in OA and NOA patients, respectively. Importantly, specific expressions of long-read transcripts were detected in germ and stomatic cells during normal spermatogenesis.
CONCLUSIONS: We have identified total full-length transcripts in OA and NOA, and new genes were found. Furthermore, specific expressed full-length transcripts were detected, and the genomic structure of transcripts was mapped in different cell types. These findings may provide valuable information on human spermatogenesis and the treatment of male infertility.
摘要：
■高通量单细胞RNA测序（scRNA-seq）广泛用于精子发生。然而,它只显示了生殖细胞和体细胞中的短读数，限制了新转录本和基因的发现。
目的：这项研究显示了梗阻性无精子症（OA）和仅支持细胞（SCO）患者精子发生的长阅读转录图。
方法：从OA和NOA患者的睾丸活检组织中分离单细胞。通过比较Pacbio长读数单细胞测序（OAn=3，NOAn=3）与短读数scRNA-seq（OAn=6，NOAn=6）来鉴定细胞培养。根据已知标记对10种生殖细胞类型和8种体细胞类型进行分类。
方法：Pacbiolong读取单细胞测序，短读scRNA-seq，聚合酶链反应。
结果：总共130,426份长读转录本（100,517份新转录本和29,909份已知转录本）和49,508份长读转录本（26,002份新转录本，和23,506已知转录物)已在OA和NOA患者中检测到，分别。此外，在OA和NOA患者中鉴定出36,373和1642个新基因，分别。重要的是,在正常精子发生过程中，在生殖细胞和气孔细胞中检测到长阅读转录本的特异性表达。
结论：我们已经确定了OA和NOA的全长转录本，发现了新的基因.此外，检测到特异性表达的全长转录本，转录本的基因组结构被定位在不同的细胞类型中。这些发现可能为人类精子发生和男性不育的治疗提供有价值的信息。
目的：这项研究显示了梗阻性无精子症（OA）和仅支持细胞（SCO）患者精子发生的长阅读转录图。
方法：从OA和NOA患者的睾丸活检组织中分离单细胞。通过比较Pacbio长读数单细胞测序（OAn=3，NOAn=3）与短读数scRNA-seq（OAn=6，NOAn=6）来鉴定细胞培养。根据已知标记对10种生殖细胞类型和8种体细胞类型进行分类。
方法：Pacbiolong读取单细胞测序，短读scRNA-seq，聚合酶链反应。
结果：总共130,426份长读转录本（100,517份新转录本和29,909份已知转录本）和49,508份长读转录本（26,002份新转录本，和23,506已知转录物)已在OA和NOA患者中检测到，分别。此外，在OA和NOA患者中鉴定出36,373和1642个新基因，分别。重要的是,在正常精子发生过程中，在生殖细胞和气孔细胞中检测到长阅读转录本的特异性表达。
结论：我们已经确定了OA和NOA的全长转录本，发现了新的基因.此外，检测到特异性表达的全长转录本，转录本的基因组结构被定位在不同的细胞类型中。这些发现可能为人类精子发生和男性不育的治疗提供有价值的信息。
