No reads after filtering error

[Seongmin-Jang-1165]

Hello BAMBU developers!!

I ran BAMBU for just quantification of my DRS data(RNA004) but the error is occured like below

I think it's because the genome and annotation file is for genome, not for transcriptome... but i still not understand about this problem

Or maybe, My input data is not appropriate for BAMBU... I used MINIMAP2 for alignment. I'll write down the code below

Could you give me some advice for this problem??

Thank you!!

[code for BAMBU]

bambuAnnotations <- prepareAnnotations("C:/Users/tjdal/OneDrive/바탕 화면/랩실/히스톤/m6A seq/m6aseq/분석/reference/GENCODE_Homo_sapiens_hg38/GRCh38.gencode.v43.basic.annotation.gtf")

se.quantOnly <- bambu(reads = "C:/Users/tjdal/OneDrive/바탕 화면/랩실/히스톤/m6A seq/NANOPORE/시퀀싱 진행_20240923/분석/minimap2/Real_Barcoded/for_dRNAseq/samtools_barcode1_sort.bam", annotations = bambuAnnotations , genome = "C:/Users/tjdal/OneDrive/바탕 화면/랩실/히스톤/m6A seq/m6aseq/분석/reference/GENCODE_Homo_sapiens_hg38/GRCh38.primary_assembly.genome.fa", discovery = FALSE)

[Code for minimap2]
/home/rnagenomics/sm/Nanopore/20240923_Histone_Direct_RNA_seq/minimap2-2.28_x64-linux/minimap2 -t 4 -ax splice -uf -k14 /home/rnagenomics/sm/Nanopore/minimap2/GRCh38.primary_assembly.transcriptome.mmi /home/rnagenomics/sm/Nanopore/20240923_Histone_Direct_RNA_seq/analysis/POD5/Real/basecalling/fastq/DORADO_barcode1.fastq > /home/rnagenomics/sm/Nanopore/20240923_Histone_Direct_RNA_seq/analysis/minimpa2/Real_Barcoded/for_dRNAseq/minimap2_barcode1.sam

samtools view -Sb minimap2_barcode1.sam > samtools_barcode1.bam

samtools sort -o samtools_barcode1_sort.bam samtools_barcode1.bam

samtools index samtools_barcode1_sort.bam &>> samtools_barcode1_sort_index.log

[cying111]

Hi @Seongmin-Jang-1165 ,

It looks to me like the bam file that you used for bambu is not appropriate. Bambu takes genome-aligned bam file instead of transcriptome-aligned bam file. My suggested alignment command is something like this (simplified):

minimap2 -t 4 -ax splice -uf -k14 GRCh38.primary_assembly.genome.fa DORADO_barcode1.fastq > minimap2_barcode1.sam

Then you proceed with samtools as what you have done before.

Let me know if your problem persists or if you need further assistance!

Thank you
Warm regards,
Ying

[Seongmin-Jang-1165]

@cying111 thanks for the advice! I regenerate BAM and there are such error, but the another error is occured..

[cying111]

Hi @Seongmin-Jang-1165 ,

Thanks for sharing the screenshot. It seems that you have successfully run bambu without error. The warnings can be ignored for now.
You may use writeBambuOutput(se.quantOnly, file = "your_save_path"), to save your output to files.
If you want to directly use your output, you may refer to here to understand the output a bit more.

Thanks and regards,
Ying

[Seongmin-Jang-1165]

@cying111 Thanks for advice! I can generate the output files.

I have additional question, the file 'uniqueCounts_transcript.txt' contains the raw read counts, not the estimated value..? I want to know the raw count of each transcript.

[cying111]

Hi @Seongmin-Jang-1165 ,

bambu reports three types of counts: counts/CPM, uniqueCounts, and fullLengthCounts

You can use counts/CPM if you want estimated counts for each transcript. For more details on how these counts are defined, see here.

Thanks and regards,
Ying