Deletions detected using Ion PGM not appearing when using Illumina

GBardai · 09-12-2019, 05:43 AM

Hello

I was wondering if anyone has encountered the issue described below or maybe able to offer some guidance:

Using the Ion PGM, Ampliseq Custom panel, we detected a heterozygous 12 bp deletion . In IgV, 150 reads have the deletion and 150 do not. This deletion was confirmed using Sanger sequencing.

When using this same sample on the NextSeq550 (Sample and library prep was tansposon based NexteraFlex for Enrichment and target enrichment was IdT xGEN research panel probe set) this deletion is seen only 2 times in 80 reads.

We attempted an alternative target enrichment set, TWIST Human Exome panel with RefSeq spike in, with the same sample and library prep system and once again came up with the same results. To rule out the sample and library prep, we are thinking to attempt this with a different enzyme fragmentation kit.

From a bioinformatics perspective, Is there a possibility that the reads having the deletions are being filtered out when the fastq files are used to generate bam?

Thanks
Ghalib

colindaven · 09-12-2019, 07:57 AM

You don't mention any bioinformatics processes used, so it is impossible to answer your question.

What you mean is during the read mapping pipeline (FASTQ-> BAM).

We use NextSeq frequently and I would expect an indel of this size to be reliably detected using either single or paired end reads (PE always better).

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09-12-2019, 05:43 AM	#1
GBardai Junior Member Location: Montreal Join Date: Sep 2019 Posts: 2	Deletions detected using Ion PGM not appearing when using Illumina Hello I was wondering if anyone has encountered the issue described below or maybe able to offer some guidance: Using the Ion PGM, Ampliseq Custom panel, we detected a heterozygous 12 bp deletion . In IgV, 150 reads have the deletion and 150 do not. This deletion was confirmed using Sanger sequencing. When using this same sample on the NextSeq550 (Sample and library prep was tansposon based NexteraFlex for Enrichment and target enrichment was IdT xGEN research panel probe set) this deletion is seen only 2 times in 80 reads. We attempted an alternative target enrichment set, TWIST Human Exome panel with RefSeq spike in, with the same sample and library prep system and once again came up with the same results. To rule out the sample and library prep, we are thinking to attempt this with a different enzyme fragmentation kit. From a bioinformatics perspective, Is there a possibility that the reads having the deletions are being filtered out when the fastq files are used to generate bam? Thanks Ghalib

09-12-2019, 07:57 AM	#2
colindaven Senior Member Location: Germany Join Date: Oct 2008 Posts: 415	You don't mention any bioinformatics processes used, so it is impossible to answer your question. What you mean is during the read mapping pipeline (FASTQ-> BAM). We use NextSeq frequently and I would expect an indel of this size to be reliably detected using either single or paired end reads (PE always better).