Thrilled to share our work on unlocking challenging specimens for @pacbio.bsky.social #HiFi sequencing, now out in Genome Biology! genomebiology.biomedcentral.com/articles/10.... We show that specimens that have been stored in ethanol for decades can still be amenable for long-read sequencing:🧵 - ThreadSky

bernhardbein.bsky.social • 17 days ago

Thrilled to share our work on unlocking challenging specimens for @pacbio.bsky.social #HiFi sequencing, now out in Genome Biology!

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-025-03487-9#Sec954

We show that specimens that have been stored in ethanol for decades can still be amenable for long-read sequencing:🧵

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adameur.bsky.social•17 days ago

This is so cool. Congrats!

bernhardbein.bsky.social•17 days ago

We managed to sequence two catfish species from ethanol-preserved specimens using the low-input protocol from @pacbio.bsky.social and assembled their genomes to a contiguitiy of 2.1 and 3.2 Mb contig N50.

However, for mammalian and reptilian samples that we tested, we could not get enough input DNA

bernhardbein.bsky.social•17 days ago

We therefore switched to the ultra-low input protocol, which contains a PCR amplification step of input DNA, employing two different polymerases we call A/B.

This way, we could sequence the maned sloth Bradypus torquatus to a coverage of 45X.

However, contig N50 of the assembly was below 1 Mb:

bernhardbein.bsky.social•17 days ago

We then used a different polymerase "C" to repeat amplification and sequencing, increasing contig N50 and compleasm/TOGA gene completness dramatically.

Aligning reads of B. torquatus to another sloth species, we saw obvious read drop-outs that polymerase C reads could cover:

bernhardbein.bsky.social•17 days ago

Combining reads of polymerase A/B and C with HiC reads, we were able to assemble the maned sloth genome to chromosome level with a contig N50 of ~4.75 Mb, a 10x increase compared to polymerase A/B reads alone.

The assemblies 3.1 Gb size also surpasses the previous 500 Mb protocol size limit.

bernhardbein.bsky.social•17 days ago

Applying polymerase A/B and C read amplification to two mollusc and one arthropod sample, we saw that assembly contiguity was either higher or comparable with polymerase C reads, and assemblies were always more contigous when combining all libraries, probably due to complementary read drop-outs.

brooklynkid53bskys.bsky.social•17 days ago

In one of the old CSH manuals, not Joy of Cloning, but maybe Bacterial Genetics by Botstein et al, they discuss long term storage of DNA and iirc, "buoyant CsCl" is touted as the best longterm storage

:)

bernhardbein.bsky.social•17 days ago

Based on our and another study, @pacbio.bsky.social has now oficially adopted polymerase C (KOD Xtreme™ Hot Start DNA polymerase) in their new Ampli-Fi input protocol:
https://www.pacb.com/blog/new-ampli-fi-ultra-low-input-protocol-will-support-hifi-sequencing-from-as-little-as-1-ng-of-dna/

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