Reply
Highlighted

Why do we have tons of NNNNNNNNNNNN reads in the FastQ files?

Posted By: sunon77, on Jan 30, 2018 at 11:16 AM

Dear 10X team, 

 

We did a 10X Chromium single-cell experiment on our Leukemia cells. We got about 7,000 cells from each lane with average 15K reads / cell. Now we want to dig into FastQ files to search for our own knock-in genes, we found tons of NNNNNNNNNNNN reads in the FastQ files. Some examples are attached here - the question is why did it happen? Are they simply poor quality reads which are stored in FastQ files.

 

 

NS500720:319:HCKTYBGX5:4:23612:6261:17366 0 Crop-Seq-BFP-Switch-hU6(Barcode)_extraction_GoldenGate_ligation_of_Crop-seq_Barcode_gRNA_BsmBI_into_Crop-Seq-BFP-Switch-hU6(BsmBI) 1471 70 60M64S * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN AAAAAEEEEEEEEEEE/EE/AEEEEEE//////666666666E66666/66666///666################################################################ RG:Z:FASTQ PL:Z:Illumina PU:ZSmiley Tongueu LB:Z:lb SM:Z:sm PG:ZSmiley FrustratedNAP NM:i:22
NS500720:319:HCKTYBGX5:4:23612:9247:17368 0 Crop-Seq-BFP-Switch-hU6(Barcode)_extraction_GoldenGate_ligation_of_Crop-seq_Barcode_gRNA_BsmBI_into_Crop-Seq-BFP-Switch-hU6(BsmBI) 1471 70 60M64S * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN AAAAAEAAEEAE<EA/<//<//A///////////66666666666666666666666666################################################################ RG:Z:FASTQ PL:Z:Illumina PU:ZSmiley Tongueu LB:Z:lb SM:Z:sm PG:ZSmiley FrustratedNAP NM:i:22
NS500720:319:HCKTYBGX5:4:23612:25370:17372 0 Crop-Seq-BFP-Switch-hU6(Barcode)_extraction_GoldenGate_ligation_of_Crop-seq_Barcode_gRNA_BsmBI_into_Crop-Seq-BFP-Switch-hU6(BsmBI) 1471 70 60M64S * 0 0 AAGCAGTGGTATCAACGCAAACTACATGGAAGGAAAAAAAAAAAAAAAAAAAAAAAAAAANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN ///AA6E66EEAEEAEEEE/E//EE6E///</E//E/EE//EE/AE/AE//AEE//E/AE################################################################ RG:Z:FASTQ PL:Z:Illumina PU:ZSmiley Tongueu LB:Z:lb SM:Z:sm PG:ZSmiley FrustratedNAP NM:i:24
NS500720:319:HCKTYBGX5:4:23612:17732:17372 0 Crop-Seq-BFP-Switch-hU6(Barcode)_extraction_GoldenGate_ligation_of_Crop-seq_Barcode_gRNA_BsmBI_into_Crop-Seq-BFP-Switch-hU6(BsmBI) 1471 70 41M1I12M1I5M64S * 0 0 AAGCAGTGGTATCAACGCAGAGTACATGGGGGGAGGCTGAGGCAGGAGAATTGCTTGAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN AAAAAEEEEEEEEEEEAEEEEEEE/EEEEEEEEEEEEEE/AEEEEEEE/EEEEAEEE<EE################################################################ RG:Z:FASTQ PL:Z:Illumina PU:ZSmiley Tongueu LB:Z:lb SM:Z:sm PG:ZSmiley FrustratedNAP NM:i:14

1 Reply

Re: Why do we have tons of NNNNNNNNNNNN reads in the FastQ files?

Posted By: Ariel, on Feb 1, 2018 at 10:49 AM

Hi!

 

It looks like your library insert size is much shorter than the sequencing read. Because your insert size is ~30bp, the sequencer then reads into the poly-A region and then the adapter. We'd be happy to help you troubleshoot this run if you email support@10xgenomics.com

 

Thanks!