Indel Puns

Just reading a paper on an algorithm which was developed to quantify indels causing frameshifts. The authors mentioned that this was needed as short read NGS has limited sensitivity for detecting indels - can't seem to understand this so would appreciate some kind explanation. Thank you!

Hello. My question is directed to those of you who have been sequencing/building pipelines to obtain COVID-19 genomes.

I have been given around 40 sequenced COVID-19 samples for which I built a pipeline to obtain the genomes. I was surprised that at the end, after quality check and filtering, none of my samples had any indels in their genomes. Is this normal, given that the samples date back to March-July 2020? Or there might be something wrong with my pipeline?

Thanks.

Hello,

I am currently trying to build a germline variant calling pipeline using GATK. One step is the Variant quality score recalibration. For this I need high confidence SNP and INDELS so I can train the model.

GATK offers these SNP and INDELS for the latest reference genome, but not for the one that I am using (GRCh37).

I read that the vcf files from the 1000genome project contains only high confident germline mutation calls and think that it might be suitable for my purpose.

So my question is, if any of you know where I could download a VCF file which contains all of the SNPs and INDELs of the phase 3 1000 genomes project.

Is this possible to download the individual chromosomes from hereand then combine them? I am afraid that this resource does not only contain "high confidence" variants. I think this might be the case, because combining these vcf files would result in a gigantic vcf file. However, the GRCh38 "gold standard high confidence snp" vcf from GATK is only 7 GB big when uncompressed.

I would be very grateful for any suggestions or links where I can download the data that I am looking for.

Cheers

Regions where deletions commonly occur are in the the N-terminal and receptor binding domains:
𝚫141-144, 𝚫189-190, 𝚫211, ins204, 𝚫256-258

https://virological.org/t/the-ongoing-evolution-of-variants-of-concern-and-interest-of-sars-cov-2-in-brazil-revealed-by-convergent-indels-in-the-amino-n-terminal-domain-of-the-spike-protein/659

So I have been planning workflow for obtaining clonal knockout cell lines. For anyone with experience doing this kind of stuff, care to comment on whether my approach seems rational?

1. Transfect pCAG-eCas9-GFP-U6-gRNA (2 sgRNA and 1 non-targeting sgRNA) using Lipofectamine 2000 (3000 if efficiency is low)
2. FACS sort approximately 2-3 million cells for 300 single GFP+ cells per sample in 96 well plates. Remaining GFP+ cells collected in a polyclonal population.
3. Culture single cell clones with conditioned media + 20% FBS.
4. Identify polyclonal NHEJ frequency by using:
   1. Guide-It Mutation Detection Kit ( https://www.takarabio.com/learning-centers/gene-function/gene-editing/crispr/cas9-knockouts/mutation-detection-kit-comparison ), a fancy mismatch cleavage assay
   2. TIDE analysis of sanger sequencing results (https://link.springer.com/protocol/10.1007/978-1-4939-9170-9_3 )
5. Polyclonal indel frequency can advise me on how many clones necessary to propagate to potentially identify homozygous KO clones. Although I understand polyclonal indel frequency is inclusive of both heterozygous and homozygous mutated alleles.

My questions are:

1. Is it advised to transfect >2 sgRNA targeting different genomic regions of the same gene together?
2. Can the PCR products obtained for the mismatch cleavage assay be used for sanger sequencing provided that the size is ~700 - 1000 bp and expected indel is ~200 bp downstream of primer.

So experts out there, does my workflow make sense? Am I missing any major considerations? Thanks for the help!

I have WGS data with reads of a certain region in the genome with a high density of short InDels. In principle, based on the mechanism whereby the InDels were created, one should be able to use these events to construct a phylogenetic tree.

What methods/software options are available for this problem?

I have a set of bacterial genomes sequenced from sputum samples of a patient with cystic fibrosis over a specific time period. We did paired end Illumina short read sequencing (151 bp) of colonies grown on selective media. I know of multiple pipelines for detection of indels and other structural variants. I am curious to hear what people typically use especially for larger variant (larger Indels/rearrangements) detection in microbial genomes. Thanks in advance! Just looking for opinions on different pipelines usage and limitations.