About pacbio's raw reads

[YilinZhang449]

Hello,
I'm a little confused, is the pacbio sequencing here generating CLR data or CCS data? How many G base of sequencing data per sample on average?

Thank you!

[aseetharam]

Hello @YilinZhang449,

We used PacBio reads. Specifically, CLR data (Subreads) was used. Please see PacBio_stats_v2.1_NAM_genomes.xlsx spreadsheet for details regarding coverage.

Thanks,

[ttian627]

Hello @YilinZhang449,

We used PacBio reads. Specifically, CLR data (Subreads) was used. Please see PacBio_stats_v2.1_NAM_genomes.xlsx spreadsheet for details regarding coverage.

Thanks,

Hello,

What's the meaning of "processed" at subreads, it seems all the pacbio CLRs passed the QC step by SequelTools.

Thank you

Tian

[DavidEHufnagel]

Hello, SequelTools calculates many statistics of sequence quantity and quality and assembles CLRs in order to do so. However, SequelTools does not generate or work with CCS data. Best, David

…

On Sat, Jan 15, 2022 at 11:24 PM YilinZhang449 ***@***.***> wrote: Hello, I'm a little confused, is the pacbio sequencing here generating CLR data or CCS data? How many G base of sequencing data per sample on average? Thank you! — Reply to this email directly, view it on GitHub <#13>, or unsubscribe <https://github.com/notifications/unsubscribe-auth/ABQPE3L53CMDDG7R2TQ6HFDUWJI7DANCNFSM5MB43XDQ> . Triage notifications on the go with GitHub Mobile for iOS <https://apps.apple.com/app/apple-store/id1477376905?ct=notification-email&mt=8&pt=524675> or Android <https://play.google.com/store/apps/details?id=com.github.android&referrer=utm_campaign%3Dnotification-email%26utm_medium%3Demail%26utm_source%3Dgithub>. You are receiving this because you are subscribed to this thread.Message ID: ***@***.***>

[aseetharam]

Hello @YilinZhang449,
We used PacBio reads. Specifically, CLR data (Subreads) was used. Please see PacBio_stats_v2.1_NAM_genomes.xlsx spreadsheet for details regarding coverage.
Thanks,

Hello,

What's the meaning of "processed" at subreads, it seems all the pacbio CLRs passed the QC step by SequelTools.

Thank you

Tian

Also, if you're referring to the spreadsheet headers, they are simplified terms for users to help understand what they are looking at: Polymerase Reads (raw) vs. Sub Reads (processed). As mentioned in the methods (supplementary), no processing was performed on subreads other than error correction itself.

Thanks,

[ttian627]

Hello @YilinZhang449,
We used PacBio reads. Specifically, CLR data (Subreads) was used. Please see PacBio_stats_v2.1_NAM_genomes.xlsx spreadsheet for details regarding coverage.
Thanks,

Hello,

What's the meaning of "processed" at subreads, it seems all the pacbio CLRs passed the QC step by SequelTools.

Thank you

Tian

I download the B73 CLRs (ERR3288278,ERR3288281,ERR3288284,ERR3288287,ERR3288290,ERR3288293,ERR3288279,ERR3288282,ERR3288285,ERR3288288,ERR3288291,ERR3288294,ERR3288280,ERR3288283,ERR3288286,ERR3288289,ERR3288292,ERR3288295), but the total bases were 195,282,635,506.
Is it right? Is it under quality control？ How?

[aseetharam]

Hi @ttian627:

Sorry, I mislabelled them. The "Raw" is subreads, and the "Processed" is Falcon error-corrected reads. If you look at the coverage stats, it uses the Subreads (labeled as "Raw") for calculations. This is true for all the tabs.
Falcon error correction was performed per the instructions provided here.

Thanks,

[ttian627]

Hello The FALOCN error corrected reads were 32x-56x coverage described in the Supplementary Material of NAM paper, but the processed reads were larger. Actually, I have the following questions. What are the downloaded data at EMBL of B73v5 (ERR3288278..etc.). The total bases were neither equal to the raw data nor the processed data. What quality control processes did they go through？quality control with smrtlink or SequelTools? Thank you Tian At 2022-04-10 00:10:52, "Arun Seetharam" ***@***.***> wrote: Hi @ttian627: Sorry, I mislabelled them. The "Raw" is subreads, and the "Processed" is Falcon error-corrected reads. If you look at the coverage stats, it uses the Subreads (labeled as "Raw") for calculations. This is true for all the tabs. Falcon error correction was performed per the instructions provided here. Thanks, — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you were mentioned.Message ID: ***@***.***>

[aseetharam]

When I sum the "Raw" reads (wrongly labeled - they are subreads), I get pretty close numbers to yours.

It could be that while downloading, you may have enabled some filtering that might have trimmed certain bases. You can calculate bases for each file and compare them to the table in the excel sheet, but I wouldn't worry about a little difference in the total count.