Not seeing Insert Size Histogram

[Mike Greenwood]

Seems like other functions are working but I don't see insert size histogram.  I'm really interested in mean/median insert size per reference.

[Konstantin Okonechnikov]

Hi!

Does BAM QC report contain insert size values in the Summary? Or is it only a figure unavailable? Typically, insert size is active only if the reads are paired-end in the input alignment BAM file.

Best regards,

   Konstantin

On Thu, May 6, 2021 at 10:13 PM Mike Greenwood <mkg...@gmail.com> wrote:

Seems like other functions are working but I don't see insert size histogram.  I'm really interested in mean/median insert size per reference.

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[Mike]

Hello!  Thank you for your reply.  It doesn't appear so, but the reads are paired.

Summary

Globals Reference size 147,124 Number of reads 704,888 Mapped reads 486,004 / 68.95% Unmapped reads 218,884 / 31.05% Mapped paired reads 486,004 / 68.95% Mapped reads, first in pair 243,009 / 34.47% Mapped reads, second in pair 242,995 / 34.47% Mapped reads, both in pair 485,990 / 68.95% Mapped reads, singletons 14 / 0% Read min/max/mean length 131 / 151 / 135.1 Duplicated reads (flagged) 105,247 / 14.93% Clipped reads 54,872 / 7.78%

ACGT Content Number/percentage of A's 15,920,580 / 25.17% Number/percentage of C's 14,334,483 / 22.66% Number/percentage of T's 17,456,214 / 27.59% Number/percentage of G's 15,252,958 / 24.11% Number/percentage of N's 6,235,280 / 9.86% GC Percentage 46.77%

Coverage Mean 472.5622 Standard Deviation 489.1574

Mapping Quality Mean Mapping Quality 0

Mismatches and indels Insertions 3,104 Mapped reads with at least one insertion 0.5% Deletions 11,941 Mapped reads with at least one deletion 1.93% Homopolymer indels 36.44%

Chromosome stats Name Length Mapped bases Mean coverage Standard deviation A*02:01:01:01 3875 2449874 632.2255 410.0818 A*02:614 3875 2264630 584.4206 399.4563 A*68:01:02:01 3875 2441390 630.0361 423.2038 A*68:164 3875 2457665 634.2361 418.559 B*35:01:01:05 4257 2005682 471.1492 209.1831 B*52:01:02:01 4257 2069348 486.1048 211.7716 C*03:03:01:01 4541 2186977 481.6069 228.3237 C*04:01:01:11 4541 2421835 533.3264 256.1043 DPA1*01:03:01:05 9834 3517406 357.6781 403.073 DPA1*01:03:01:05X 9834 3483382 354.2182 399.2769 DPB1*04:02:01:02 11615 7757044 667.8471 741.2827 DQA1*03:01:01:01 6767 4281604 632.7182 357.1965 DQA1*04:01:01:07 6767 3960401 585.2521 323.2718 DQB1*03:02:01:01 7781 5680592 730.0594 556.4607 DQB1*04:02:01:04 7781 5511543 708.3335 542.3835 DRB1*04:11:01 18509 3296625 178.1093 264.6255 DRB1*08:02:01:01 18509 5854153 316.2868 450.6595 DRB4*01:03:01:01 16631 7885090 474.12 639.4515

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[Konstantin Okonechnikov]

Hi!

Since the insert size is not in statistics, it means that it's either not present in read alignment records ( but this is not suitable since 1st and 2nd reads were found, not sure why it could be like this), or all of insert size them appears to be less than zero e.g reads within the pair are overlapping.  

Would be curious to check the BAM file or a subsample from it e.g. either from one chromosome only or small random ( http://okko73313.blogspot.com/2013/03/random-subsample-from-bam-file.html  ) 

Best regards,

   Konstantin 

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[Mike]

Grabbed 10 pairs.  The bottom pair has an overlap of 4 and the second to last has a gap of 56.

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[Konstantin Okonechnikov]

By some reason the insert size is zero in all of the read alignments. Simply it's the column 9 in SAM format, just in case here's example overview: https://accio.github.io/bioinformatics/2020/03/10/filter-bam-by-insert-size.html 

Did you compute your values (-4,56) manually? Which tool was applied for alignment?  The insert sizes are expected to be provided already in output BAM/SAM file.

Best regards,

   Konstantin

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[Mike]

I'm working on HLA typing and the kit we use comes with software for analysis of fastq data.  The software releases the BAM file and also has a tool to view the alignments.  It is in this tool I can see the gap/overlap.  It is quite slow to open each sample to review the read lengths, though, so maybe I can have the developers include an informative TLEN value.  Thanks for your help.

Mike

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