<p>I have a read from BAM file as following</p>
<pre><code>NB501288:541:HCWKVBGXB:2:21107:25304:16421  0   #16 41910661    255 27M167903N19M266888N9M  *   0   0   TTTCTTTTTTTTTTTTTTTTTTTTTTGAAAGGGGGTTTTTTTGTTAAACCAAGGG array('B', [32, 32, 32, 32, 32, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 14, 14, 14, 14, 14, 14, 14, 36, 32, 14, 36, 36, 36, 36, 14, 32, 32, 14, 14, 14, 14, 14, 14, 14, 27, 14, 14, 14, 14, 14])    [('NH', 1), ('HI', 1), ('AS', 40), ('nM', 1), ('RE', 'I'), ('xf', 0), ('li', 0), ('CR', 'CATTTCACAGGGACTA'), ('CY', 'AAAAAEEEEEEEEEEE'), ('CB', 'CATTTCACAGGGACTA-1'), ('UR', 'AATTTACAGC'), ('UY', 'EEEEAEEEEE'), ('UB', 'AATTTACAGC'), ('RG', 'Batch1:0:1:HCWKVBGXB:2')]
</code></pre>
<p>From cigar information, I noticed that there is a very larg gap between 3 partial aligned parts in the same read (167903N and 266888N). And I also observed the same behavior in most of reads that mapped to the same interested region.</p>
<p>I'm trying to understand the reason behind this behavior. I run fastqc on the files and some of metrics failed (over-represented sequence (polyA), adaptor content, base quality) in only some files (not all). Will those bad quality affect the alignment performance? As I know, those adapter and polyA are trimmed by cellranger, and also those low quality bases are discarded by aligner as well. Or maybe I misunderstand something here.</p>
<p>Is there anyone met this problem before or have any idea about the reason for it?
Thanks for any support.</p>

CC BY-SA 4.0
2024-05-02T10:22:11.763Z
Have you considered that you are seeing a large insertion? Are you sure this is a problem and not a structural variant?
terdon
CC BY-SA 4.0
2024-05-02T11:44:43.560Z
Wildly guessing that this is chr16 in mm10, I see a gene with short exons and looooong introns in the genome browser at these coordinates. Can this simply be splicing?!
ATpoint
CC BY-SA 4.0
2024-05-02T12:16:21.180Z
@terdon I supposed it's not a variant since it happens in all samples (WT and KO)
Tien
CC BY-SA 4.0
2024-05-02T12:23:01.370Z
Please [edit] your question and give us more details. What kind of sequencing? What chemistry? WES? WGS? Panel? Short read? Long read? Amplicon? Capture? What species are you looking at? What genome are you aligning against? Are you sequencing individuals or pooling samples? How many samples? How many have this feature? We need details to be able to help well.
terdon
CC BY-SA 4.0
2024-05-02T12:24:54.607Z
@ATpoint Since this read was not assigned to any valid gene ID, I already checked and found that each match mapped to different geneID.
Tien
CC BY-SA 4.0
2024-05-02T12:37:40.087Z
What I know about the data is:
- 10x Genomics and hashtag libraries were prepared as per standard 10x Genomics Chromium Single Cell 3’ Reagent Kits v.3 and MULTI-Seq27 protocols
- snRNA-Seq libraries
Tien
CC BY-SA 4.0
2024-05-02T16:32:22.067Z
Please ***edit*** this into your question. You mention single cell, is this single cell DNA sequencing? The most important detail is still what kind of analysis this is, what did you sequence? Whole genome? Whole exome? Gene panel? And what species? I can see that this is a highly repetitive, low complexity region, is that expected? Can you show us an image of the alignment opened in IGV or a similar tool and focused on the region of interest?
terdon
<phylogenetics><scrnaseq><sam><cellranger>
Large skip after aligning using Cellranger
# Large skip after aligning using Cellranger

(Asked by: username_0 on 2024-05-02)

I have a read from BAM file as following
NB501288:541:HCWKVBGXB:2:21107:25304:16421  0   #16 41910661    255 27M167903N19M266888N9M  *   0   0   TTTCTTTTTTTTTTTTTTTTTTTTTTGAAAGGGGGTTTTTTTGTTAAACCAAGGG array('B', [32, 32, 32, 32, 32, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 36, 14, 14, 14, 14, 14, 14, 14, 36, 32, 14, 36, 36, 36, 36, 14, 32, 32, 14, 14, 14, 14, 14, 14, 14, 27, 14, 14, 14, 14, 14])    [('NH', 1), ('HI', 1), ('AS', 40), ('nM', 1), ('RE', 'I'), ('xf', 0), ('li', 0), ('CR', 'CATTTCACAGGGACTA'), ('CY', 'AAAAAEEEEEEEEEEE'), ('CB', 'CATTTCACAGGGACTA-1'), ('UR', 'AATTTACAGC'), ('UY', 'EEEEAEEEEE'), ('UB', 'AATTTACAGC'), ('RG', 'Batch1:0:1:HCWKVBGXB:2')]

From cigar information, I noticed that there is a very larg gap between 3 partial aligned parts in the same read (167903N and 266888N). And I also observed the same behavior in most of reads that mapped to the same interested region.
I'm trying to understand the reason behind this behavior. I run fastqc on the files and some of metrics failed (over-represented sequence (polyA), adaptor content, base quality) in only some files (not all). Will those bad quality affect the alignment performance? As I know, those adapter and polyA are trimmed by cellranger, and also those low quality bases are discarded by aligner as well. Or maybe I misunderstand something here.
Is there anyone met this problem before or have any idea about the reason for it?
Thanks for any support.

**Tags:** phylogenetics, scrnaseq, sam, cellranger
> **Comments:**
> **username_1**: Have you considered that you are seeing a large insertion? Are you sure this is a problem and not a structural variant?  
> **username_2**: Wildly guessing that this is chr16 in mm10, I see a gene with short exons and looooong introns in the genome browser at these coordinates. Can this simply be splicing?!  
> **username_0**: @terdon I supposed it's not a variant since it happens in all samples (WT and KO)  
> **username_1**: Please [edit] your question and give us more details. What kind of sequencing? What chemistry? WES? WGS? Panel? Short read? Long read? Amplicon? Capture? What species are you looking at? What genome are you aligning against? Are you sequencing individuals or pooling samples? How many samples? How many have this feature? We need details to be able to help well.  
> **username_0**: @ATpoint Since this read was not assigned to any valid gene ID, I already checked and found that each match mapped to different geneID.  
> **username_0**: What I know about the data is: - 10x Genomics and hashtag libraries were prepared as per standard 10x Genomics Chromium Single Cell 3’ Reagent Kits v.3 and MULTI-Seq27 protocols - snRNA-Seq libraries  
> **username_1**: Please ***edit*** this into your question. You mention single cell, is this single cell DNA sequencing? The most important detail is still what kind of analysis this is, what did you sequence? Whole genome? Whole exome? Gene panel? And what species? I can see that this is a highly repetitive, low complexity region, is that expected? Can you show us an image of the alignment opened in IGV or a similar tool and focused on the region of interest?  

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## 0 answers

