Quality control of the full-length transcriptomes
The FL transcriptomes for R. a. hainanus, R. a. himalayanus and Myotis ricketti were constructed based on sequencing data of three separated libraries on the PacBio Sequel platform. Specifically, a total of 3,444,947 subreads with 6,448,987,299 nucleotides, 3,255,638 subreads with 6,504,282,447 nucleotides and 3,403,451 subreads with 7,190,237,257 nucleotides were generated for R. a. hainanus, R. a. himalayanus and Myotis ricketti respectively. After quality control, we obtained 137,159 circular consensus sequencing (CCS) reads for R. a. hainanus, 137,160 CCS reads for R. a. himalayanus and 152,251 CCS reads for Myotis ricketti. With the standard IsoSeq. 3 classification and clustering pipeline, we identified 111,806 FLNC for R. a. hainanus, 105,713 FLNC for R. a. himalayanus and 122,222 FLNC for Myotis ricketti. After isoform-level polishing, 10384, 9984 and 10932 high quality isoforms were retained in R. a. hainanus, R. a. himalayanus and Myotis ricketti respectively. After removing redundancy with CD-HIT-EST and filtering isoforms shorter than 200 bp, the final FL transcriptomes for R. a. hainanus, R. a. himalayanus and Myotis ricketti (FL-CF-Rhai, FL-CF-Rhim and FL-FM-Myo, respectively) contain 10103, 9676 and 10504 FL isoforms with an average length of 2251, 2370 and 2530 bp, respectively (Table 2). Finally, the FL transcriptome from both CF and FM bats (FL-CF-FM) contains 26,342 transcripts with an average length of 2,405 bp (Table 2). BUSCO analysis revealed that a total of 2,354 (57.4%) BUSCOs were included in FL-CF-FM. We also found 39.9%, 38.1% and 41.9% BUSCOs in FL-CF-Rhai, FL-CF-Rhim and FL-FM-Myo, respectively (Table 4). Given the highly specialized function of the cochlea, we should not expect a high level of BUSCO value in FL transcriptome of cochlea. A recent single cell RNA-seq study has identified a similar number of genes expressed in the murine cochlea (a total of 12,944)30.
Quality control of annotation
Four FL transcriptomes (FL-CF-Rhai, FL-CF-Rhim, FL-FM-Myo, and FL-CF-FM) were functionally annotated by performing DIAMOND and BLASTx searches against the Nr and UniProt databases separately. For FL-CF-FM, 24,793 and 24,198 transcripts were annotated by Nr database and UniProt database, respectively (Table 3). After combining the annotation results from the two databases, a total of 24,833 transcripts were annotated in at least one database. We obtained similar annotation results for FL-CF-Rhai, FL-CF-Rhim and FL-FM-Myo (Table 3). Transcripts without annotations might be novel isoforms of echolocating animals or due to the lack of representative sequences for cochlea in public databases.
Source: Ecology - nature.com
