Please help!! Completely new to pre-processing Drop-Seq SRA file from database

TofuKaj · 08-24-2020, 02:34 AM

Dear all great helpers,

I'm completely new to Drop-seq analysis, and I do have no clue how to preprocess Drop-Seq data acquired from SRA database. For example, I have tried downloading and spiting SRR11014434 data file. Now I have two fastq files as follows: SRR11014434_1.fastq and SRR11014434_2.fastq.

I've started by learning from the Drop-seq cookbook (https://github.com/broadinstitute/Dr...t_Cookbook.pdf). As far as I understand the SRR11014434_2.fastq file contains data about the UMI and Cell barcode, while the SRR11014434_1.fastq file contains expression data of the cell.

Since the cookbook demonstrate step-by-step starting from raw read counts acquired from sequencer, I'm now totally confused with what I'm doing. Some of the very basic but critical questions are as follows:

1) Is the data already demultiplexed?
2) Am I supposed to align SRR11014434_2.fastq by STAR? What should I do after this step? The cookbook talked about merging mapped and unmapped BAM file, tagging exon/gene annotations. But I totally how am I suppose to do this without the unmapped bam file (All I could think about is trying to include unmaaped read the output sam file acquired from STAR using --outSAMunmapped Within option and use it as the merged file).

Please help me!!

Kaj

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08-24-2020, 02:34 AM	#1
TofuKaj Member Location: Bangkok, Thailand Join Date: Apr 2015 Posts: 10	Please help!! Completely new to pre-processing Drop-Seq SRA file from database Dear all great helpers, I'm completely new to Drop-seq analysis, and I do have no clue how to preprocess Drop-Seq data acquired from SRA database. For example, I have tried downloading and spiting SRR11014434 data file. Now I have two fastq files as follows: SRR11014434_1.fastq and SRR11014434_2.fastq. I've started by learning from the Drop-seq cookbook (https://github.com/broadinstitute/Dr...t_Cookbook.pdf). As far as I understand the SRR11014434_2.fastq file contains data about the UMI and Cell barcode, while the SRR11014434_1.fastq file contains expression data of the cell. Since the cookbook demonstrate step-by-step starting from raw read counts acquired from sequencer, I'm now totally confused with what I'm doing. Some of the very basic but critical questions are as follows: 1) Is the data already demultiplexed? 2) Am I supposed to align SRR11014434_2.fastq by STAR? What should I do after this step? The cookbook talked about merging mapped and unmapped BAM file, tagging exon/gene annotations. But I totally how am I suppose to do this without the unmapped bam file (All I could think about is trying to include unmaaped read the output sam file acquired from STAR using --outSAMunmapped Within option and use it as the merged file). Please help me!! Kaj