Sequencing Human/Mouse ATAC libraries together?

ETBA · 08-03-2020, 09:15 AM

Hi all,

I am a novice to this and currently preparing mouse and human ATAC libraries. All samples are on different barcodes and we want to compare these samples, so we think it's better to sequence (100 bp, paired end, HiSeq-X-Ten) everything together to get similar depth across all and reduce batch effects.

I'm unsure if this is a problem for the sequencer or for the analysis too? After read-trimming and FastQC check, when I go to align the samples with BWA-MEM or Bowtie2 for example, could I run one analysis and select for aligned human and mouse reads or do two separate runs targeted to 1 species to separate them out?

Any help / suggestions are much appreciated.

Thanks, Eric

GenoMax · 08-03-2020, 09:57 AM

As long as you use unique indexes for all samples (dual indexes would be best), QC and then balance the libraries in the pool so the contents are more or less uniform, you can mix and sequence these together. Post-run demultiplexing will separate all of your samples into independent files on a per sample basis.

ETBA · 08-03-2020, 10:21 AM

That's great, thanks for the information. Just to be safe, i'm assuming that the dual indexes are also the oligos specified in Buenrostro, 2013?

GenoMax · 08-03-2020, 01:37 PM

Unique dual indexes are used in library prep. See this.

ETBA · 08-03-2020, 04:47 PM

Brilliant that was very helpful. Although a bit of shame as many samples are already done on a single indexing strategy with i7 so i'll have to stick with that. I'll certainly consider the use of a dual indexing strategy next time.
Thanks again!

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08-03-2020, 09:15 AM	#1
ETBA Junior Member Location: UK Join Date: Sep 2018 Posts: 4	Sequencing Human/Mouse ATAC libraries together? Hi all, I am a novice to this and currently preparing mouse and human ATAC libraries. All samples are on different barcodes and we want to compare these samples, so we think it's better to sequence (100 bp, paired end, HiSeq-X-Ten) everything together to get similar depth across all and reduce batch effects. I'm unsure if this is a problem for the sequencer or for the analysis too? After read-trimming and FastQC check, when I go to align the samples with BWA-MEM or Bowtie2 for example, could I run one analysis and select for aligned human and mouse reads or do two separate runs targeted to 1 species to separate them out? Any help / suggestions are much appreciated. Thanks, Eric

08-03-2020, 09:57 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	As long as you use unique indexes for all samples (dual indexes would be best), QC and then balance the libraries in the pool so the contents are more or less uniform, you can mix and sequence these together. Post-run demultiplexing will separate all of your samples into independent files on a per sample basis.

08-03-2020, 10:21 AM	#3
ETBA Junior Member Location: UK Join Date: Sep 2018 Posts: 4	That's great, thanks for the information. Just to be safe, i'm assuming that the dual indexes are also the oligos specified in Buenrostro, 2013?

08-03-2020, 01:37 PM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Unique dual indexes are used in library prep. See this.

08-03-2020, 04:47 PM	#5
ETBA Junior Member Location: UK Join Date: Sep 2018 Posts: 4	Brilliant that was very helpful. Although a bit of shame as many samples are already done on a single indexing strategy with i7 so i'll have to stick with that. I'll certainly consider the use of a dual indexing strategy next time. Thanks again!